Methods of treating neuroendocrine tumors using frizzled-binding agents

ABSTRACT

Novel methods of treating neuroendocrine tumors are provided. In one embodiment, the method comprises administering to a subject in need thereof a therapeutically effective dose of a Wnt antagonist. In one embodiment, the Wnt antagonist is an anti-FZD antibody. In another embodiment, the Wnt antagonist is a soluble FZD receptor polypeptide. In a further embodiment, the Wnt antagonist is an anti-Wnt antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/059,918, filed Oct. 22, 2013, which claims priority benefit of U.S.Provisional Application No. 61/717,294, filed Oct. 23, 2012, and U.S.Provisional Application No. 61/760,529, filed Feb. 4, 2013, each ofwhich is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The field of this invention generally relates to methods of treatingneuroendocrine tumors. In one embodiment, the method comprisesadministering to a subject in need thereof a therapeutically effectivedose of a Wnt antagonist.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2293_0950003_SeqListing_ST25.txt; Size: 190 kilobytes; and Date ofCreation: Jan. 6, 2016) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich—breast, lung, colorectal, and prostate—account for over half ofall new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

The Wnt signaling pathway has been identified as a potential target forcancer therapy. The Wnt signaling pathway is one of several criticalregulators of embryonic pattern formation, post-embryonic tissuemaintenance, and stem cell biology. More specifically, Wnt signalingplays an important role in the generation of cell polarity and cell fatespecification including self-renewal by stem cell populations.Unregulated activation of the Wnt pathway is associated with numeroushuman cancers where it can alter the developmental fate of tumor cellsto maintain them in an undifferentiated and proliferative state. Thuscarcinogenesis can proceed by usurping homeostatic mechanismscontrolling normal development and tissue repair by stem cells (reviewedin Reya & Clevers, 2005, Nature 434:843; Beachy et al., 2004, Nature432:324).

The Wnt signaling pathway was first elucidated in the Drosophiladevelopmental mutant wingless (wg) and from the murine proto-oncogeneint-1, now Wnt1 (Nusse & Varmus, 1982, Cell 31:99-109; Van Ooyen &Nusse, 1984, Cell 39:233-40; Cabrera et al., 1987, Cell 50:659-63;Rijsewijk et al., 1987, Cell 50:649-57). Wnt genes encode secretedlipid-modified glycoproteins of which 19 have been identified inmammals. These secreted ligands activate a receptor complex consistingof a Frizzled (Fzd) receptor family member and low-density lipoprotein(LDL) receptor-related protein 5 or 6 (LPRS/6). The Fzd receptors areseven transmembrane domain proteins of the G-protein coupled receptor(GPCR) superfamily and contain a large extracellular N-terminal ligandbinding domain with 10 conserved cysteines, known as a cysteine-richdomain (CRD) or Fri domain. There are ten human FZD receptors: FZD1-10.Different Fzd CRDs have different binding affinities for specific Wnts(Wu & Nusse, 2002, J. Biol. Chem. 277:41762-9), and Fzd receptors havebeen grouped into those that activate the canonical β-catenin pathwayand those that activate non-canonical pathways described below (Milleret al., 1999, Oncogene 18:7860-72). To form the receptor complex thatbinds the FZD ligands, FZD receptors interact with LRP5/6, single passtransmembrane proteins with four extracellular EGF-like domainsseparated by six YWTD amino acid repeats (Johnson et al., 2004, J. BoneMineral Res. 19:1749).

The Wnt/beta-catenin signaling pathway has been implicated in thedevelopment of gastrointestinal carcinoid tumors. Fujimori et al.,Cancer Res. 61(18): 6656-9 (2001). Nuclear translocation of β-cateninprotein but absence of β-catenin and APC mutation in gastrointestinalcarcinoid tumor has also been observed. Su et al., Ann. Surg. Oncol.13(12): 1604-9 (2006). 72 cases of gastrointestinal carcinoid tumor wereinvestigated both immunohistochemically and by direct sequencing ofbeta-catenin. Accumulation of beta-catenin in the cytoplasm and/ornucleus was observed in 57 cases (79.2%). Mutations were also detectedin exon 3 of beta-catenin in 27 cases (37.5%), and in APC in one case(1.4%). Su et al. also reported the investigation of 91 gastrointestinalcarcinoid tumors and, for comparison, 26 extragastrointestinal carcinoidtumors by immunohistochemical detection of beta-catenin protein anddirect sequencing of exon 3 of the beta-catenin gene and exon 15 of theAPC gene. Cytoplasmic accumulation and/or nuclear translocation ofbeta-catenin were found in 27 gastrointestinal carcinoid tumors (29.7%)but not in any extragastrointestinal carcinoid tumors. Neitherbeta-catenin nor APC gene mutation was detected in any of the cases withnuclear expression of beta-catenin.

SUMMARY OF THE INVENTION

The present invention provides methods of treating a neuroendocrinetumor. Thus in one aspect, the invention provides methods of inhibitingthe growth of a neuroendocrine tumor, comprising contacting theneuroendocrine tumor with an effective amount of a Wnt antagonist. Inanother aspect, the invention provides methods of inhibiting theproliferation of neuroendocrine tumor cells, comprising contacting theneuroendocrine tumor cells with an effective amount of a Wnt antagonist.In another aspect, the invention provides methods of reducing thetumorigenicity of neuroendocrine tumor cells, comprising contacting theneuroendocrine tumor cells with an effective amount of a Wnt antagonist.In another aspect, the invention provides methods of inducingneuroendocrine tumor cells to differentiate, comprising contacting theneuroendocrine tumor cells with an effective amount of a Wnt antagonist.In another aspect, the invention provides methods of inhibiting thegrowth of a neuroendocrine tumor, comprising administering to a subjectin need thereof a therapeutically effective amount of a Wnt antagonist.In another aspect, the invention provides methods of inhibiting theproliferation of neuroendocrine tumor cells, comprising administering toa subject in need thereof a therapeutically effective amount of a Wntantagonist. In another aspect, the invention provides methods oftreating neuroendocrine cancer, comprising administering to a subject inneed thereof a therapeutically effective amount of a Wnt antagonist. Incertain embodiments the subject is a human subject.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the neuroendocrine tumor is a low grade, medium grade,or high grade neuroendocrine tumor. In further embodiments, theneuroendocrine tumor is a functional neuroendocrine tumor or anon-functional neuroendocrine tumor. In further embodiments, theneuroendocrine tumor is selected from the group consisting ofgastroenteropancreatic neuroendocrine tumor, carcinoid tumor,pheochromocytoma, paraganglioma, medullary thyroid cancer, pulmonaryneuroendocrine tumor and thymic neuroendocrine tumor. In furtherembodiments, the neuroendocrine tumor is a carcinoid tumor or apancreatic neuroendocrine tumor.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the Wnt antagonist is an antibody. In furtherembodiments, the Wnt antagonist is an antibody that specifically bindsto at least one human Wnt. In further embodiments, the Wnt antagonist isan antibody that specifically binds to at least one human frizzledreceptor (FZD). In further embodiments, the Wnt antagonist is a solubleFZD receptor.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the Wnt antagonist is an antibody that specificallybinds to at least one human frizzled receptor (FZD). In furtherembodiments, the antibody specifically binds to the extracellular domainof at least one human FZD. In further embodiments, the antibodyspecifically binds to a human FZD selected from the group consisting ofFZD1, FZD2, FZD5, FZD7, and FZD8. In further embodiments, the antibodyspecifically binds to FZD7. In further embodiments, the antibodyspecifically binds to more than one human FZD. In further embodiments,the antibody specifically binds to three or more human FZD selected fromthe group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. In furtherembodiments, the antibody specifically binds to more than one human FZDselected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8.In further embodiments, the antibody specifically binds to FZD1, FZD2,FZD5, FZD7, and FZD8.

In further embodiments, the antibody blocks ligand binding to FZD. Infurther embodiments, the antibody blocks Wnt binding to FZD. In furtherembodiments, the antibody blocks the activation of FZD.

In further embodiments, the antibody comprises: (1) a heavy chain CDR1comprising GFTFSHYTLS (SEQ ID NO:31), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:32), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:33); and/or (2) (a) a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:34), a light chain CDR2 comprising DKSNRPSG (SEQID NO:35), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:36);or (b) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:41), a lightchain CDR2 comprising EKDNRPSG (SEQ ID NO:42), and a light chain CDR3comprising SSFAGNSLE (SEQ ID NO:43). In further embodiments, theantibody comprises: a VH comprising the amino acid sequence of SEQ IDNO:37; and/or a VL comprising the amino acid sequence of SEQ ID NO:38 or44. In further embodiments, the antibody comprises: a heavy chaincomprising the amino acid sequence of SEQ ID NO:39; and/or a light chaincomprising the amino acid sequence of SEQ ID NO:40 or 45.

In further embodiments, the antibody is a monoclonal antibody. Infurther embodiments, the antibody is a recombinant antibody, a chimericantibody, a humanized antibody, a human antibody, or an antibodyfragment. In further embodiments, the antibody is a monospecificantibody or a bispecific antibody. In further embodiments, the antibodyis an IgA, IgD, IgE, IgG or IgM antibody. In further embodiments, theantibody is an IgG1 or IgG2 antibody.

In further embodiments, the Wnt antagonist is OMP-18R5 (also known as“vantictumab”).

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the Wnt antagonist is a soluble FZD receptor. Infurther embodiments, the soluble FZD receptor binds to Wnt. In furtherembodiments, the soluble receptor comprises a fragment of theextracellular domain of a human FZD receptor. In further embodiments,the fragment of the extracellular domain of the human FZD receptorcomprises the Fri domain of the human FZD receptor.

In further embodiments, the human FZD receptor is selected from thegroup consisting of FZD4, FZD5, and FZD8. In further embodiments, thehuman FZD receptor is FZD8. In further embodiments, the FZD8 Fri domaincomprises the amino acid sequence of SEQ ID NO:28.

In further embodiments, the soluble receptor further comprises a humanFc domain. In further embodiments, the human Fc domain comprises theamino acid sequence of SEQ ID NO:95.

In further embodiments, the Wnt antagonist is OMP-54F28.

In certain embodiments of each of the aforementioned aspects orembodiments, as well as other aspects and/or embodiments describedelsewhere herein, the methods further comprise contacting the tumor ortumor cells with a second therapeutic agent, or administering a secondtherapeutic agent to the subject. In further embodiments, the secondtherapeutic agent is a chemotherapeutic agent. In further embodiments,the second therapeutic agent is a kinase inhibitor, somatostatin analogor an mTOR pathway inhibitor. In further embodiments, the secondtherapeutic agent is sunitinib, octreotide, or everolimus. In furtherembodiments, the second therapeutic agent is an antibody. In furtherembodiments, the second therapeutic agent is an angiogenesis inhibitor.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A-C. Effect of Wnt inhibitors on neuroendocrine tumor growth. Thesize of tumor lesions in a pancreatic neuroendocrine tumor patient wasreduced following the administration of the OMP-18R5 anti-FZD7 antibody.(FIG. 1A) Radiographic assessment of the size of target and non-targetlesions at day 56 and day 112 of OMP-18R5 anti-FZD7 antibody treatment.BL denotes the baseline size of the lesions before the administration ofOMP-18R5. (FIG. 1B) CT image of the tumor lesions before (Baseline) andafter 112 days of OMP-18R5 administration. (FIG. 1C) CT image of thetumor lesions before (Baseline) and after 112 days of OMP-18R5administration. The tumor lesion at day 112 displays radiologic signs ofcalcification.

FIG. 2. Days on study for patients on OMP-18R5 Phase 1a study. Thenumber of days each of the patients (n=18) enrolled in the OMP-18R5Phase 1a study has stayed on the study as of Jan. 25, 2013, is showngraphically in the figure. Arrows indicate the patients who remained onthe study as of Jan. 25, 2013. The vertical lines indicate dates oftumor assessments on the study. The neuroendocrine tumor patients arepatients 003 (Patient 3 in Example 1), 010 (Patient 10 in Example 1),and 012 (Patient 12 in Example 1). The other patients on the study hadother types of advanced solid tumors such as colorectal cancer, breastcancer, melanoma, and pancreatic cancer.

FIG. 3. Days on study for patients with neuroendocrine tumors onOMP-18R5 Phase la study were compared to days on treatment with priorregimens. Patient 10, a 69-year-old woman with neuroendocrine tumor ofthe pancreas, continues on study with stable disease for 279 days (as ofJan. 25, 2013). Patient 12, a 77-year-old woman with carcinoid,continues on study with stable disease for 210 days (as of Jan. 25,2013).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of inhibiting the growth of aneuroendocrine tumor, methods of inhibiting proliferation ofneuroendocrine tumor cells, methods of treating a neuroendocrine cancer,methods of inhibiting neuroendocrine tumor metastases, methods ofinducing neuroendocrine tumor cell differentiation, methods of reducingtumorgenicity of neuroendocrine tumor cells and methods of reducing thefrequency of cancer stem cells or tumor initiating cells in aneuroendocrine tumor. In some embodiments, the methods provided hereincomprise administering a Wnt antagonist to a subject. In someembodiments, the Wnt antagonist is a FZD-binding agent that specificallybinds to one or more human FZD receptors. In further embodiments, theFZD-binding agent is an antibody that specifically binds to one or bindsto one or more human FZD receptors. In some embodiments, the Wntantagonist is a Wnt binding agent that specifically binds to one or morehuman Wnt polypeptide. In some embodiments, the Wnt binding agent is asoluble FZD receptor. In some embodiments, the Wnt binding agent is ananti-Wnt antibody.

Human patients with late stage neuroendocrine tumors were treated withlow doses of the

OMP-18R5 anti-FZD antibody in the context of a Phase 1 clinical trialfor patients with late stage solid tumors. (Example 1.) Surprisingly,one of the patients (a patient having a pancreatic neuroendocrine tumor)showed a reduction in tumor lesion size after 112 days of treatment withOMP-18R5 and remained on study without evidence of any progression ofdisease for 279 days (as of Jan. 25, 2013). Additionally, newcalcification was seen in one of the patient's lesions which mayrepresent possible signs of tumor cell necrosis and/or differentiation.In addition, two patients with neuroendocrine tumors having carcinoidhistology were also able to stay on the study for surprisingly longperiods of time with stable disease during treatment with OMP-18R5.(Example 1.) Collectively, these results suggest that OMP-18R5 may beparticularly useful in the treatment of a variety of neuroendocrinetumors.

1. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “antagonist” is used herein to include any molecule thatpartially or fully blocks, inhibits, or neutralizes the expression of orthe biological activity of a protein, (e.g., a cancer stem cell marker).The blocking, inhibiting, and/or neutralizing of biological activityincludes, but is not limited to, inhibition of tumor growth. The term“antagonist” also includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of the Wnt pathway. Theterm “Wnt antagonist” is used herein to include any molecule thatpartially or fully blocks, inhibits or neutralizes the signaling of theWnt pathway (e.g., canonical Wnt signaling), or partially or fullyblocks, inhibits or neutralizes a biological activity of a component ofthe Wnt pathway. Wnt antagonists do not necessarily bind Wnt. Forinstance, in certain embodiments Wnt antagonists bind one or more othercomponents of the Wnt pathway such as one or more FZD receptors.Suitable Wnt antagonist molecules include, but are not limited to,fragments and/or amino acid sequence variants of native FZD receptorproteins including soluble FZD receptors, as well as derivatives ofsoluble Frizzled-related proteins (SFRPs), and derivatives of Rorproteins. Suitable Wnt antagonist molecules further include, but are notlimited to, antibodies that specifically bind to one or more FZDreceptors and antibodies that specifically bind to one or more Wntpolypeptide. Soluble SFRP and Ror receptors are described in US Pat.Appl. Pub. No. 2011/0305695, which is herein incorporated by reference.

In vivo and in vitro assays for determining whether an agent (e.g.,soluble FZD receptor or anti-FZD antibody) inhibits Wnt signaling areknown in the art. For example, cell-based, luciferase reporter assaysutilizing a TCF/Luc reporter vector containing multiple copies of theTCF-binding domain upstream of a firefly luciferase reporter gene may beused to measure canonical Wnt signaling levels in vitro (Gazit et al.,1999, Oncogene 18; 5959-66). The level of Wnt signaling in the presenceof one or more Wnts (e.g., Wnt(s) expressed by transfected cells orprovided by Wnt-conditioned media) with the agent present is compared tothe level of signaling without the agent present. In addition to theTCF/luc reporter assay, the effect of an agent (e.g., soluble FZDreceptor or anti-FZD antibody) on canonical Wnt signaling can bemeasured in vitro or in vivo by measuring the effect of the agent on thelevel of expression of beta-catenin regulated genes, such as c-myc (Heet al., Science, 281:1509-12 (1998)), cyclin D1 (Tetsu et al., Nature,398:422-6 (1999)) and/or fibronectin (Gradl et al. Mol. Cell Biol.,19:5576-87 (1999)). In certain embodiments, the effect of the agent onWnt signaling can also be assessed by measuring the effect of the agenton the phosphorylation state of Dishevelled-1, Dishevelled-2,Dishevelled-3, LRP5, LRP6, and/or beta-catenin. In still furtherembodiments, the effect of the agent on Wnt signaling is determined byassessing the impact of the agent on the expression level of one or moregenes in a Wnt signature. Non-limiting examples of the use of suchassays to assess inhibition of canonical Wnt signaling are disclosed inU.S. Pat. Appl. Pub. No. 2012/0027778, which is incorporated byreference herein in its entirety.

As used herein the term “soluble receptor” refers to an amino-terminalextracellular fragment of a receptor protein preceding the transmembranedomain that can be secreted from a cell in soluble form. In someembodiments, the receptor protein is a FZD receptor. In someembodiments, the receptor protein is the ROR1 or ROR2 receptor. Incertain embodiments, the soluble receptor is linked in-frame with apolypeptide that increases the half-life of the soluble receptor. Incertain embodiments, the polypeptide that increases half-life is a humanFc domain.

As used herein the term “FZD soluble receptor” refers to anamino-terminal extracellular fragment of a human FZD receptor proteinpreceding the transmembrane domain of the receptor that can be secretedfrom a cell in soluble form. FZD soluble receptors comprising the entireamino-terminal extracellular domain (ECD) (referred to herein as “FZDECD”) as well as smaller fragments of the ECD are envisioned. FZDsoluble receptors comprising the Fri domain (referred to herein as “FZDFri”) are also disclosed. Soluble FZD receptors are described in US Pat.Appl. Pub. No. 2011/0305695, which is herein incorporated by reference.

FZD Fri soluble receptors can demonstrate altered biological activity,(e.g., increased protein half-life) compared to soluble receptorscomprising the entire FZD ECD. Protein half-life can be furtherincreased by covalent modification with polyethylene glycol (PEG) orpolyethylene oxide (PEO). FZD soluble receptors include FZD ECD or Fridomains linked in-frame to other functional and structural proteinsincluding, but not limited to, a human Fc region (e.g., human Fc derivedfrom immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, orIgM); protein tags (e.g., myc, FLAG, GST); other endogenous proteins orprotein fragments; or any other useful protein sequence including anylinker region between a FZD ECD or Fri domain and a linked protein. Incertain embodiments, the Fri domain of a FZD receptor is directly linkedto a human Fc region. In certain embodiments, the Fri domain of a FZDreceptor is linked to human IgG1 Fc (referred to herein as “FZD Fri.Fc,”e.g. “FZD8 Fri.Fc”). In some embodiments, the Fri domain of a FZDreceptor is linked to a human Fc region with a peptide linker. FZDsoluble receptors also include variant proteins comprising amino acidinsertions, deletions, substitutions, and/or conservative substitutions.

As used herein, the term “linker” or “linker region” refers to a linkerinserted between a first polypeptide (e.g., a FZD component) and asecond polypeptide (e.g., an Fc region). In some embodiments, the linkeris a peptide linker. Linkers should not adversely affect the expression,secretion, or bioactivity of the polypeptides. Preferably, linkers arenot antigenic and do not elicit an immune response.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature,321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen etal., 1988, Science, 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

That a polypeptide or other agent (e.g., antibody or soluble receptor)“specifically binds” to a protein means that the polypeptide or otheragent reacts or associates more frequently, more rapidly, with greaterduration, with greater affinity, or with some combination of the aboveto the protein than with alternative substances, including unrelatedproteins. In certain embodiments, “specifically binds” means, forinstance, that an agent (e.g., antibody or soluble receptor) binds to aprotein with a K_(D) of about 0.1 mM or less, but more usually less thanabout 1 μM. In certain embodiments, “specifically binds” means that anagent (e.g., antibody or soluble receptor) binds to a protein at timeswith a K_(D) of at least about 0.1 μM or less, at least about 0.01 μM orless, and at other times at least about 1 nM or less. Because of thesequence identity between homologous proteins in different species,specific binding can include an agent (e.g., antibody or solublereceptor) that recognizes a particular protein such as a Wnt protein ora frizzled receptor in more than one species. Likewise, because ofhomology between different paralogues (e.g., the different human Wntproteins or human frizzled proteins) in certain regions of theirsequences, specific binding can include a polypeptide or an agent (e.g.,antibody or soluble receptor) that recognizes more than one paralogue(e.g., more than one human Wnt protein or more than one human frizzledprotein). It is understood that an agent (e.g., antibody or solublereceptor) that specifically binds to a first target may or may notspecifically bind to a second target. As such, “specific binding” doesnot necessarily require (although it can include) exclusive binding,i.e. binding to a single target. Thus, an agent (e.g., antibody orsoluble receptor) may, in certain embodiments, specifically bind to morethan one target (e.g., multiple different human Wnt proteins or multipledifferent frizzled proteins, such as FZD1, FZD2, FZD5, FZD7, and/orFZD8). In certain embodiments, the multiple targets of an antibody maybe bound by the same antigen-binding site on the antibody. For example,an antibody may, in certain instances, comprise two identicalantigen-binding sites, each of which specifically binds two or morehuman frizzled receptors (e.g., human FZD1, FZD2, FZD5, FZD7, and/orFZD8). In certain alternative embodiments, an antibody may be bispecificand comprise at least two antigen-binding sites with differingspecificities. By way of non-limiting example, a bispecific antibody maycomprise one antigen-binding site that recognizes an epitope on onefrizzled receptor, such as human FZD5, and further comprises a second,different antigen-binding site that recognizes a different epitope on asecond frizzled receptor, such as human FZD8. Generally, but notnecessarily, reference to binding means specific binding.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. The term cancer is understoodto encompass Wnt-dependent cancers. Examples of cancer include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

“Tumor” and “neoplasm” refer to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

The terms “cancer stem cell,” “tumor stem cell,” or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; (2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells,” “tumor stem cells,” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” refers to the functional features of a solidtumor stem cell including the properties of self-renewal (giving rise toadditional tumorigenic cancer stem cells) and proliferation to generateall other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor. These properties of self-renewal and proliferation to generateall other tumor cells confer on cancer stem cells the ability to formpalpable tumors upon serial transplantation into an immunocompromisedmouse compared to non-tumorigenic tumor cells, which are unable to formtumors upon serial transplantation. It has been observed thatnon-tumorigenic tumor cells may form a tumor upon primarytransplantation into an immunocompromised mouse after obtaining thetumor cells from a solid tumor, but those non-tumorigenic tumor cells donot give rise to a tumor upon serial transplantation.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

The term “therapeutically effective amount” refers to an amount of anagent (e.g., antibody, soluble receptor, polypeptide, polynucleotide,small organic molecule, or other drug) effective to “treat” a disease ordisorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the agent can reduce the number ofcancer cells; reduce the tumor size; inhibit or stop cancer cellinfiltration into peripheral organs including, for example, the spreadof cancer into soft tissue and bone; inhibit and stop tumor metastasis;inhibit and stop tumor growth; relieve to some extent one or more of thesymptoms associated with the cancer; reduce morbidity and mortality;improve quality of life; decrease tumorigenicity, tumorigenic frequency,or tumorigenic capacity of a tumor; reduce the number or frequency ofcancer stem cells in a tumor; differentiate tumorigenic cells to anon-tumorigenic state; or a combination of such effects. To the extentthe agent prevents growth and/or kills existing cancer cells, it can bereferred to as cytostatic and/or cytotoxic.

As used herein the term “inhibit tumor growth” refers to any mechanismby which tumor cell growth can be inhibited. In certain embodiments,tumor cell growth is inhibited by slowing proliferation of tumor cells.In certain embodiments, tumor cell growth is inhibited by haltingproliferation of tumor cells. In certain embodiments, tumor cell growthis inhibited by killing tumor cells. In certain embodiments, tumor cellgrowth is inhibited by inducing apoptosis of tumor cells. In certainembodiments, tumor cell growth is inhibited by inducing differentiationof tumor cells. In certain embodiments, tumor cell growth is inhibitedby depriving tumor cells of nutrients. In certain embodiments, tumorcell growth is inhibited by preventing migration of tumor cells. Incertain embodiments, tumor cell growth is inhibited by preventinginvasion of tumor cells.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorigenic frequency, or tumorigeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

2. Methods of Treatment

The present invention provides methods of treating neuroendocrinetumors. Neuroendocrine tumors (NETs) are tumors that arise from cells ofthe endocrine (hormonal) and nervous systems. Neuroendocrine tumors(NETs) include a group of tumors with a range of morphologic,functional, and behavioral characteristics. These tumors are generallyslow growing and behave in an indolent fashion. However, they have thepotential to spread, primarily to the liver, and when they do, they canbe life threatening and difficult to treat with current modalities.

Neuroendocrine tumors are classified by the site of their origin. Incertain embodiments, the NET is selected from the group consisting ofpancreatic neuroendocrine tumors (pNETs) and carcinoid tumors of thelung, stomach, duodenum, jejunum, ileum, colon and rectum. In furtherembodiments, the NET is selected from the group consistingneuroendocrine tumors of the ovary, thymus, thyroid medulla, adrenalglands (e.g., pheocromocytoma) and paraganglia (paraganglioma). Incertain embodiments, the NET treated by the methods described herein issmall cell lung cancer (SCLC). In certain alternative embodiments, theNET is not small cell lung cancer. In certain embodiments, NETs arepancreatic neuroendocrine tumors (PETs) or carcinoid tumors. In certainembodiments, the NET is not small cell lung cancer, a pancreatic cancer,or a thyroid cancer.

Neuroendocrine tumors are also classified by grade and differentiation.See, e.g., Phan et al., Pancreas, 39(6):784-798 (2012). In certainembodiments, the neuroendocrine tumor is a well differentiated, lowgrade tumor. In certain embodiments, the neuroendocrine tumor is amoderately differentiated, intermediate grade tumor. In certainembodiments, the neuroendocrine tumor is a poorly differentiated, highgrade tumor. In one embodiment, low grade tumors are characterized by <2mitoses per 10 HPF (high power fields) and no necrosis. In oneembodiment, intermediate grade tumors are characterized by 2-10 mitosesper 10 HPF (high power fields) or foci of necrosis. In one embodiment,high grade tumors are characterized by >10 mitoses per 10 HPF (highpower fields).

Neuroendocrine tumors are also classified as functional andnon-functional NETs. NETs are considered functional when a specificclinical syndrome is induced due to excessive production of hormones bythe tumor cells. Examples of functional NETs include, but are notlimited to, carcinoid tumors, which can result in carcinoid syndrome,and functional pNETs, for example, insulinomas, gastrinomas, vasoactiveintestinal peptide (VIP)omas, glucagonomas, and somatostatinomas.Non-functional NETs are not associated with a clinical syndrome due toexcessive production of hormones by the tumor cells, but can stillproduce symptoms related to the presence of the tumor or its metastasis(e.g., abdominal pain or bloating). In certain embodiments, theneuroendocrine tumor is a functional NET. In certain embodiments, theneuroendocrine tumor is a non-functional NET. In certain embodiments,the neuroendocrine tumor is selected from the group consisting offunctional carcinoid tumor, insulinoma, gastrinoma, vasoactiveintestinal peptide (VIP)oma, glucagonoma, serotoninoma, histaminoma,ACTHoma, pheocromocytoma, and somatostatinoma. In certain embodiments,the neuroendocrine tumor is not SCLC.

In certain embodiments, the neuroendocrine tumor is a primary tumor. Incertain embodiments, the neuroendocrine tumor is metastatic tumor. Incertain embodiments, the neuroendocrine tumor has not spread outside ofthe wall of the primary organ. In certain embodiments, theneuroendocrine tumor has spread through the wall of the primary organand to nearby tissues, such as fat, muscle, or lymph nodes. In certainembodiments, the neuroendocrine tumor has spread to tissues or organsaway from the primary organ, for example, to the liver, bones, or lungs.

In certain embodiments, the neuroendocrine cancer or tumor is refractoryto treatment. As a non-limiting example, the cancer or tumor may bechemorefractory (i.e., resistant to one or more forms of chemotherapy).In certain embodiments, the cancer or tumor is resistant to treatmentwith a somatostatin analog. In certain embodiments, the cancer or tumoris resistant to treatment with a kinase inhibitor.

In certain embodiments, the neuroendocrine cancer or tumor hasmetastasized to the liver. By way of non-limiting example, theneuroendocrine cancer or tumor is a carcinoid or pancreaticneuroendocrine tumor that has metastasized to the liver.

In one aspect, the present invention provides the use of a Wntantagonist (e.g., an anti-FZD antibody or soluble FZD receptor) in thetreatment of neuroendocrine tumor. In certain embodiments, the Wntantagonist is useful for inhibiting Wnt signaling (e.g., canonical Wntsignaling) in a neuroendocrine tumor cell, inhibiting neuroendocrinetumor growth, inducing neuroendocrine tumor differentiation, reducingneuroendocrine tumor volume, and/or reducing the tumorigenicity of aneuroendocrine tumor. The methods of use can be in vitro, ex vivo, or invivo methods. In certain embodiments, the Wnt antagonist is the antibodyOMP-18R5. In certain embodiments, the Wnt antagonist is the solublereceptor OMP-54F28.

The present invention provides for methods of treating neuroendocrinetumor comprising administering a therapeutically effective amount of aWnt antagonist (e.g., an anti-FZD antibody or soluble FZD receptor) to asubject (e.g., a subject in need of treatment). In certain embodiments,the neuroendocrine tumor is a pancreatic neuroendocrine tumor. Incertain embodiments, the neuroendocrine tumor is a carcinoid. In certainembodiments, the neuroendocrine tumor is neuroendocrine tumor of thelung. By way of non-limiting example, the neuroendocrine tumor in thelung may be SCLC. In certain embodiments, the neuroendocrine tumor isnot SCLC. In certain embodiments, the subject is a human. In certainembodiments, the Wnt antagonist is OMP-18R5. In certain embodiments, theWnt antagonist is OMP-54F28.

The present invention further provides methods for inhibitingneuroendocrine tumor growth using the Wnt antagonists (e.g., anti-FZDantibodies and soluble FZD receptors) described herein. In certainembodiments, the method of inhibiting the neuroendocrine tumor growthcomprises contacting the tumor cell with a Wnt antagonist (e.g., ananti-FZD antibody or soluble FZD receptor) in vitro. For example, animmortalized neuroendocrine tumor cell line is cultured in medium towhich is added the Wnt antagonist (e.g., an anti-FZD antibody or solubleFZD receptor) to inhibit tumor growth. In some embodiments,neuroendocrine tumor cells are isolated from a patient sample such as,for example, a tissue biopsy, pleural effusion, or blood sample andcultured in medium to which is added a Wnt antagonist (e.g., an anti-FZDantibody or soluble FZD receptor) to inhibit tumor growth. In certainembodiments, the Wnt antagonist is OMP-18R5. In certain embodiments, theWnt antagonist is OMP-54F28.

In some embodiments, the method of inhibiting neuroendocrine tumorgrowth comprises contacting the neuroendocrine tumor or tumor cells withthe Wnt antagonist (e.g., an anti-FZD antibody or soluble FZD receptor)in vivo. In certain embodiments, contacting a neuroendocrine tumor ortumor cell with a Wnt antagonist (e.g., an anti-FZD antibody or solubleFZD receptor) is undertaken in an animal model. For example, a Wntantagonist (e.g., an anti-FZD antibody or soluble FZD receptor) may beadministered to neuroendocrine tumor xenografts that have been grown inimmunocompromised mice (e.g. NOD/SCID mice) to inhibit neuroendocrinetumor growth. In some embodiments, neuroendocrine tumor cancer stemcells are isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice that are then administered a Wnt antagonist(e.g., an anti-FZD antibody or soluble FZD receptor) to inhibitneuroendocrine tumor cell growth. In some embodiments, the Wntantagonist (e.g., anti-FZD antibody or soluble FZD receptor) isadministered at the same time or shortly after introduction oftumorigenic cells into the animal to prevent neuroendocrine tumorgrowth. In some embodiments, the Wnt antagonist (e.g., anti-FZD antibodyor soluble FZD receptor) is administered as a therapeutic after thetumorigenic cells have grown to a specified size. In certainembodiments, the Wnt antagonist is OMP-18R5. In certain embodiments, theWnt antagonist is OMP-54F28.

In certain embodiments, the method of inhibiting neuroendocrine tumorgrowth comprises administering to a subject a therapeutically effectiveamount of a Wnt antagonist (e.g., an anti-FZD antibody or soluble FZDreceptor). In certain embodiments, the subject is a human. In certainembodiments, the subject has a neuroendocrine tumor or has had a tumorremoved.

In certain embodiments, the neuroendocrine tumor is a tumor in which Wntsignaling is active. In certain embodiment, the Wnt signaling that isactive is canonical Wnt signaling. In certain embodiments, theneuroendocrine tumor is a Wnt-dependent tumor. For example, in someembodiments, the tumor is sensitive to axin over-expression. In certainembodiments, the tumor does not comprise an inactivating mutation (e.g.,a truncating mutation) in the adenomatous polyposis coli (APC) tumorsuppressor gene or an activating mutation in the beta-catenin gene. Incertain embodiments, the tumor expresses one or more genes in a Wnt genesignature, i.e., one or more genes up-regulated or down-regulated by theWnt signaling pathway. In certain embodiments, the neuroendocrine tumorfor which a subject is being treated involves such a tumor.

In certain embodiments, the neuroendocrine tumor expresses one or morehuman frizzled receptors to which the Wnt antagonist FZD-bindingantibody described herein binds. In certain embodiments, theneuroendocrine tumor overexpresses the human frizzled receptor(s). Incertain embodiments, the Wnt antagonist is OMP-18R5.

In certain embodiments, the neuroendocrine tumor expresses one or morehuman Wnt polypeptides to which the Wnt antagonist soluble FZD receptordescribed herein binds. In certain embodiments, the neuroendocrine tumoroverexpresses the human Wnt polypeptide(s). In certain embodiments, theWnt antagonist is OMP-54F28.

In certain embodiments, the neuroendocrine tumor expresses one or morehuman Wnt polypeptides to which the Wnt antagonist anti-Wnt antibodydescribed herein binds. In certain embodiments, the neuroendocrine tumoroverexpresses the human Wnt polypeptide(s).

In certain embodiments, the neuroendocrine tumor is a pancreaticneuroendocrine tumor. In certain embodiments, the neuroendocrine tumoris a carcinoid. In certain embodiments, the neuroendocrine tumor isneuroendocrine tumor of the lung. In certain embodiments, theneuroendocrine tumor is not SCLC.

The invention also provides a method of inhibiting Wnt signaling in aneuroendocrine tumor cell comprising contacting the cell with aneffective amount of a Wnt antagonist (e.g., an anti-FZD antibody orsoluble FZD receptor). In certain embodiments, the method is an in vivomethod wherein the step of contacting the cell with the Wnt antagonist(e.g., anti-FZD antibody or soluble FZD receptor) comprisesadministering a therapeutically effective amount of the Wnt antagonistto the subject. In some alternative embodiments, the method is an invitro or ex vivo method. In certain embodiments, the Wnt signaling thatis inhibited is canonical Wnt signaling. In certain embodiments, the Wntsignaling is signaling by Wnt1, Wnt2, Wnt3, Wnt3A, Wnt7a, Wnt7b, and/orWnt10B. In certain embodiments, the Wnt signaling is signaling by Wnt1,Wnt3A, Wnt7b, and/or Wnt10B.

In addition, the invention provides a method of reducing thetumorigenicity of a neuroendocrine tumor in a subject, comprisingadministering a therapeutically effective amount of a Wnt antagonist(e.g., an anti-FZD antibody or soluble FZD receptor) to the subject. Incertain embodiments, the neuroendocrine tumor comprises cancer stemcells. In certain embodiments, the frequency of cancer stem cells in theneuroendocrine tumor is reduced by administration of the agent. Incertain embodiments, the Wnt antagonist is OMP-18R5. In certainembodiments, the Wnt antagonist is OMP-54F28.

Thus, the invention also provides a method of reducing the frequency ofcancer stem cells in a neuroendocrine tumor, comprising contacting thetumor with an effective amount of a Wnt antagonist (e.g., an anti-FZDantibody or soluble FZD receptor).

The invention further provides methods of differentiating tumorigenicneuroendocrine tumor cells into non-tumorigenic cells comprisingcontacting the tumorigenic neuroendocrine tumor cells with a Wntantagonist (e.g., an anti-FZD antibody or soluble FZD receptor) byadministering the Wnt antagonist to a subject that has a neuroendocrinetumor comprising the tumorigenic cells or that has had such aneuroendocrine tumor removed.

The use of the Wnt antagonists (e.g., an anti-FZD antibodies and solubleFZD receptors) described herein to induce the differentiation ofneuroendocrine tumor cells is also provided. For example, methods ofinducing cells to differentiate comprising contacting the cells with aneffective amount of a Wnt antagonist (e.g., an anti-FZD antibody orsoluble FZD receptor) described herein are envisioned. Methods ofinducing cells in a neuroendocrine tumor in a subject to differentiatecomprising administering a therapeutically effective amount of a Wntantagonist (e.g., an anti-FZD antibody or soluble FZD receptor) to thesubject are also provided. In certain embodiments, the differentiationof neuroendocrine tumor cells is associated with changes in theradiographic image of the tumor lesion. In certain embodiments, thedifferentiation of neuroendocrine tumor cells is associated withcalcification in the tumor lesion. In certain embodiments, the Wntantagonist is OMP-18R5. In certain embodiments, the Wnt antagonist isOMP-54F28.

Methods of treating a neuroendocrine tumor in a subject, wherein theneuroendocrine tumor is associated with Wnt signaling activation and/oris characterized by an increased level of stem cells and/or progenitorcells are further provided. In some embodiments, the treatment methodscomprise administering a therapeutically effective amount of a Wntantagonist (e.g., an anti-FZD antibody or soluble FZD receptor) to thesubject. In certain embodiments, the Wnt signaling is canonical Wntsignaling

In certain embodiments, in addition to administering the Wnt antagonist(e.g., anti-FZD antibody or soluble FZD receptor) described herein, themethod or treatment further comprises administering a second anti-canceragent (prior to, concurrently with, and/or subsequently toadministration of the Wnt antagonist). Pharmaceutical compositionscomprising the Wnt antagonist and the second anti-cancer agent are alsoprovided. In certain embodiments, the administration of the combinationof the Wnt antagonist and a second anti-cancer agent has a synergisticeffect, such as a synergistic effect on the frequency of cancer stemcells.

It will be appreciated that the combination of a Wnt antagonist (e.g.,anti-FZD antibody or soluble FZD receptor) and a second anti-canceragent may be administered in any order or concurrently. In selectedembodiments, the Wnt antagonist will be administered to patients thathave previously undergone treatment with the second anti-cancer agent.In certain other embodiments, the Wnt antagonist and the secondanti-cancer agent will be administered substantially simultaneously orconcurrently. For example, a subject may be given the Wnt antagonistwhile undergoing a course of treatment with the second anti-cancer agent(e.g., chemotherapy). In certain embodiments, the Wnt antagonist will beadministered within 1 year of the treatment with the second anti-canceragent. In certain alternative embodiments, the Wnt antagonist will beadministered within 10, 8, 6, 4, or 2 months of any treatment with thesecond anti-cancer agent. In certain other embodiments, the Wntantagonist will be administered within 4, 3, 2, or 1 week of anytreatment with the second anti-cancer agent. In some embodiments, theWnt antagonist will be administered within 5, 4, 3, 2, or 1 days of anytreatment with the second anti-cancer agent. It will further beappreciated that the two agents or treatment may be administered to thesubject within a matter of hours or minutes (i.e., substantiallysimultaneously).

Useful classes of anti-cancer agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, performing compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like. In certain embodiments, the secondanti-cancer agent is an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor.

Anticancer agents that may be administered in combination with the Wntantagonists (e.g., anti-FZD antibodies or soluble FZD receptors) includechemotherapeutic agents. Thus, in some embodiments, the method ortreatment involves the combined administration of a Wnt antagonist and achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with a Wnt antagonist can occur priorto, concurrently with, or subsequent to administration ofchemotherapies. Chemotherapies contemplated by the invention includechemical substances or drugs which are known in the art and arecommercially available, such as gemcitabine, irinotecan, doxorubicin,5-fluorouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide,thiotepa, busulfan, cytoxin, TAXOL, methotrexate, cisplatin, melphalan,vinblastine and carboplatin. Combined administration can includeco-administration, either in a single pharmaceutical formulation orusing separate formulations, or consecutive administration in eitherorder but generally within a time period such that all active agents canexert their biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992).

Chemotherapeutic agents useful in the instant invention also include,but are not limited to, alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan,improsulfan, and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and me thylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and antiandrogens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In certain embodiments, the chemotherapeutic agent is a kinaseinhibitor. In certain embodiments, the kinase inhibitor is amulti-targeted receptor tyrosine kinase inhibitor. Kinase inhibitorsinclude, but are not limited to, sunitinib (marketed as Sutent byPfizer), pazopanib, crizotinib, dasatinib. In certain embodiments, thesecond anticancer agent is sunitinib.

In certain embodiments, the chemotherapeutic agent is an inhibitor ofmammalian target of rapamycin (mTOR). mTOR inhibitors include, but arenot limited to, temsirolimus, sirolimus, deforolimus and everolimus. Incertain embodiments, the second anticancer agent is everolimus.

In certain embodiments, the chemotherapeutic agent is a somatostatinanalog. Somatostatin analogs act through interaction with specific, highaffinity membrane receptors for somatostatin. Somatostatin analogsinclude, but are not limited to, octreotide, somatulin, and RC 160(octastatin). In certain embodiments, the second anticancer agent isoctreotide.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCL, daunorubicin citrate, mitoxantrone HCL, actinomycin D,etoposide, Topotecan HCL, teniposide (VM-26), and irinotecan. In certainembodiments, the second anticancer agent is irinotecan.

In certain embodiments, the chemotherapeutic agent is an alkylatingagent. In certain embodiments, the chemotherapeutic agent istemozolomide.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,THIOGUANINE (GlaxoSmithKline), 5-azacytidine, 6-mercaptopurine,azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, andcladribine, as well as pharmaceutically acceptable salts, acids, orderivatives of any of these. In certain embodiments, the secondanticancer agent is gemcitabine. In certain embodiments, the tumor to betreated is a pancreatic neuroendocrine tumor and the second anticanceragent is an anti-metabolite (e.g., gemcitabine).

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin By way ofnon-limiting example, the agent comprises a taxane. In certainembodiments, the agent comprises paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the agent is paclitaxel (TAXOL),docetaxel (TAXOTERE), albumin-bound paclitaxel (e.g., ABRAXANE),DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments,the antimitotic agent comprises a vinca alkaloid, such as vincristine,binblastine, vinorelbine, or vindesine, or pharmaceutically acceptablesalts, acids, or derivatives thereof. In some embodiments, theantimitotic agent is an inhibitor of Eg5 kinesin or an inhibitor of amitotic kinase such as Aurora A or Plkl.

In certain embodiments, the treatment involves the combinedadministration of a Wnt antagonist (e.g., anti-FZD antibody or solubleFZD receptor) described herein and radiation therapy. Treatment with theWnt antagonist can occur prior to, concurrently with, or subsequent toadministration of radiation therapy. Any dosing schedule for suchradiation therapy can be used as determined by the skilled practitioner.

In some embodiments, the second anti-cancer agent comprises an antibody.Thus, treatment can involve the combined administration of a Wntantagonist (e.g., anti-FZD antibody or soluble FZD receptor) withantibodies against tumor-associated antigens including, but not limitedto, antibodies that bind to EGFR, ErbB2, HER2, DLL4, Notch and/or VEGF.Exemplary, anti-DLL4 antibodies, are described, for example, in U.S.Patent Application Publication No. US 2008/0187532, incorporated byreference herein in its entirety. In certain embodiments, the secondanti-cancer agent is an antibody that is an angiogenesis inhibitor(e.g., an anti-VEGF antibody). Additional anti-DLL4 antibodies aredescribed in, e.g., International Patent Publication Nos. WO 2008/091222and WO 2008/0793326, and U.S. Patent Application Publication Nos. US2008/0014196, US 2008/0175847, US 2008/0181899, and US 2008/0107648,each of which is incorporated by reference herein in its entirety.Exemplary anti-Notch antibodies are described, for example, in U.S.Patent Application Publication No. US 2008/0131434, incorporated byreference herein in its entirety. In certain embodiments, the secondanti-cancer agent is an antibody that is an angiogenesis inhibitor(e.g., an anti-VEGF antibody). In certain embodiments, the secondanti-cancer agent is an inhibitor of Notch signaling. In certainembodiments, the second anti-cancer agent is AVASTIN (Bevacizumab),Herceptin (Trastuzumab), VECTIBIX (Panitumumab), or Erbitux (Cetuximab).Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

Furthermore, treatment can include administration of one or morecytokines (e.g., lymphokines, interleukins, tumor necrosis factors,and/or growth factors) or can be accompanied by surgical removal ofcancer cells or any other therapy deemed necessary by a treatingphysician.

For the treatment of the disease, the appropriate dosage of a Wntantagonist (e.g., anti-FZD antibody or soluble FZD receptor) describedherein depends on the type of neuroendocrine tumor to be treated, theseverity and course of the neuroendocrine tumor, the responsiveness ofthe neuroendocrine tumor, whether the Wnt antagonist (e.g., anti-FZDantibody or soluble FZD receptor) is administered for therapeutic orpreventative purposes, previous therapy, patient's clinical history, andso on all at the discretion of the treating physician. The Wntantagonist (e.g., anti-FZD antibody or soluble FZD receptor) can beadministered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the neuroendocrine tumor is achieved (e.g. reduction intumor size). Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body of the patient and willvary depending on the relative potency of an individual Wnt antagonist(e.g., anti-FZD antibody or soluble FZD receptor). The administeringphysician can easily determine optimum dosages, dosing methodologies andrepetition rates. In certain embodiments, dosage is from 0.01 μg to 100mg per kg of body weight, and can be given once or more daily, weekly,monthly or yearly. In certain embodiments, the Wnt antagonist (e.g.,anti-FZD antibody or soluble FZD receptor) is given once every two weeksor once every three weeks. In certain embodiments, the dosage of the Wntantagonist (e.g., anti-FZD antibody or soluble FZD receptor) is fromabout 0.1 mg to about 20 mg per kg of body weight. The treatingphysician can estimate repetition rates for dosing based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. In certain embodiments, the Wnt antagonist is OMP-18R5. Incertain embodiments, the Wnt antagonist is OMP-54F28.

In certain embodiments, OMP-18R5 is administered intravenously at a doseof about 0.1 mg/kg to about 20 mg/kg or a dose of about 0.5 mg/kg toabout 10 mg/kg. Such doses may, in some embodiments, be given aboutevery week, every two weeks, every three weeks or every four weeks. Incertain embodiments, OMP-18R5 is administered intravenously at a dosageof about 0.5 mg/kg to about 10 mg/kg about every two to four weeks. Incertain embodiments, OMP-18R5 is administered intravenously at a dosageof about 1.0 mg/kg to about 10 mg/kg approximately about every threeweeks. In certain embodiments, OMP-18R5 is administered intravenously ata dosage of (a) at least about 0.5 mg/kg about every one to two weeks or(b) at least about 1.0 mg/kg about every three weeks. In certainembodiments, the antibody is administered at a dosage of about 0.5 mg/kgto about 1.0 mg/kg about every one to two weeks. In some alternativeembodiments, the antibody is administered at a dosage of about 1.0 mg/kgto about 5.0 mg/kg about every three weeks.

By way of non-limiting example, OMP-54F28 may be administeredintravenously at a dose of about 0.1 mg/kg to about 20 mg/kg. This dosemay, in some embodiments, be given every week, every two weeks, everythree weeks or every four weeks. In certain embodiments, OMP-54F28 isadministered intravenously at a dosage of about 0.5 mg/kg to about 10mg/kg every two to four weeks. In certain embodiments, OMP-54F28 isadministered intravenously at a dosage of about 0.5 mg/kg to about 10mg/kg about every three weeks.

3. FZD-Binding Agents

Another aspect of the methods of the invention is the use of aFZD-binding agent (e.g., anti-FZD antibody) in the treatment ofneuroendocrine tumors. In certain embodiments, the FZD-binding agents(e.g., anti-FZD antibodies) that are useful in the methods of theinvention specifically bind one or more human frizzled receptors (FZDs).In certain embodiments, the agents specifically bind two, three, four,five, six, seven, eight, nine, or ten frizzled receptors. The humanfrizzled receptor or receptors bound by the agent can be selected fromthe group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8,FZD9, and FZD10. In certain embodiments, the one or more human frizzledreceptors comprise FZD1, FZD2, FZD5, FZD7, and/or FZD8. In certainembodiments, the one or more human frizzled receptors comprise FZD7. Incertain embodiments, the one or more human frizzled receptors compriseFZD5 and/or FZD8. In certain embodiments, the agent specifically bindsFZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, theFZD-binding agent specifically binds FZD7. In certain embodiments, theFZD-binding agent specifically binds FZD5. The full-length amino acid(aa) and nucleotide (nt) sequences for FZD1-10 are known in the art andalso provided herein as SEQ ID NO:1 (FZD1 aa), SEQ ID NO:2 (FZD2 aa),SEQ ID NO:3 (FZD3 aa), SEQ ID NO:4 (FZD4 aa), SEQ ID NO:5 (FZD5 aa), SEQID NO:6 (FZD6 aa), SEQ ID NO:7 (FZD7 aa), SEQ ID NO:8 (FZD8 aa), SEQ IDNO:9 (FZD9 aa), SEQ ID NO:10 (FZD10 aa).

In certain embodiments, a FZD-binding agent (e.g., anti-FZD antibody)that is useful in the methods of the invention specifically binds to twoor more human frizzled receptors. In certain embodiments, the two ormore human frizzled receptors are selected from the group consisting ofFZD2, FZD5, FZD7, and FZD8. In certain embodiments, the two or morefrizzled receptors comprise FZD 1 and a second frizzled receptorselected from the group consisting of FZD2, FZD5, FZD7, and FZD8. Incertain embodiments, the two or more frizzled receptors comprise FZD2and a second frizzled receptor selected from the group consisting ofFZD1, FZD5, FZD7, and FZD8. In certain embodiments, the two or morefrizzled receptors comprise FZD5 and a second frizzled receptor selectedfrom the group consisting of FZD1, FZD2, FZD7, and FZD8. In certainembodiments, the two or more frizzled receptors comprise both FZD5 andFZD8. In certain embodiments, the two or more frizzled receptorscomprise FZD7 and a second frizzled receptor selected from the groupconsisting of FZD1, FZD2, FZD5, and FZD8. In certain embodiments, theagent specifically binds to three or more human frizzled receptors. Incertain embodiments, the three or more human frizzled receptors comprisethree or more frizzled receptors selected from the group consisting ofFZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the agentfurther specifically binds to one or more additional human frizzledreceptors.

In certain embodiments, a FZD-binding agent (e.g., anti-FZD antibody)that is useful in the methods of the invention specifically binds to theextracellular domain (ECD) within the one or more human frizzledreceptors to which it binds. Sequences of the extracellular domain ofeach of the human frizzled receptors are known in the art and are alsoprovided as SEQ ID NO:11 (FZD1 ECD), SEQ ID NO:12 (FZD2 ECD), SEQ IDNO:13 (FZD3 ECD), SEQ ID NO:14 (FZD4 ECD), SEQ ID NO:15 (FZD5 ECD), SEQID NO:16 (FZD6 ECD), SEQ ID NO:17 (FZD7 ECD), SEQ ID NO:18 (FZD8 ECD),SEQ ID NO:19 (FZD9 ECD), and SEQ ID NO:20 (FZD10 ECD). Particularlyuseful antibodies are described in U.S. Pat. No. 7,982,013 and U.S. Pat.Appl. Pub. No. 2012/0027778, which are herein incorporated by referencein their entirety.

In certain embodiments, a FZD-binding agent (e.g., anti-FZD antibody)that is useful in the methods of the invention specifically binds to theFri domain (FRI) (also known as the cysteine-rich domain (CRD)) withinthe human frizzled receptor(s) to which it binds. Sequences of the Fridomain of each of the human frizzled receptors are known in the art andare also provided herein. The Fri domain of FZD1 includes approximatelyamino acids 87-237 of SEQ ID NO:11. The Fri domain of FZD2 includesapproximately amino acids 24-159 of SEQ ID NO:12. The Fri domain of FZD3includes approximately amino acids 23-143 of SEQ ID NO:13. The Fridomain of FZD4 includes approximately amino acids 40-170 of SEQ IDNO:14. The Fri domain of FZD5 includes approximately amino acids 27-157of SEQ ID NO:15. The Fri domain of FZD6 includes approximately aminoacids 19-146 of SEQ ID NO:16. The Fri domain of FZD7 includesapproximately amino acids 33-170 of SEQ ID NO:17. The Fri domain of FZD8includes approximately amino acids 28-158 of SEQ ID NO:18. The Fridomain of FZD9 includes approximately amino acids 23-159 of SEQ IDNO:19. The Fri domain of FZD10 includes approximately amino acids 21-154of SEQ ID NO:20. The corresponding, predicted Fri domains for each ofthe human FZD receptors are provided as SEQ ID NOs:21-30. The minimal,core Fri domain sequences for each of the human FZD receptors (FZD1-10)are provided as SEQ ID NOs:73-82. Those of skill in the art may differin their understanding of the exact amino acids corresponding to thevarious Fri domains. Thus in specific embodiments, the N-terminus orC-terminus of the domains outlined above and herein can extend or beshortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

In certain embodiments, an individual antigen-binding site of aFZD-binding antibody is capable of binding (or binds) the one, two,three, four, or five (or more) human frizzled receptors. In certainembodiments, an individual antigen-binding site of the FZD-bindingantibody is capable of specifically binding one, two, three, four, orfive human frizzled receptors selected from the group consisting ofFZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, an individualbinding site of the antibody specifically binds to at least FZD5 andFZD8.

In certain embodiments, a FZD-binding agent (e.g., anti-FZD antibody)that is useful in the methods of the invention binds to one or more (forexample, two or more, three or more, or four or more) human frizzledreceptors with a dissociation constant (K_(D)) of about 1 μM or less,about 100 nM or less, about 40 nM or less, about 20 nM or less, or about10 nM or less. For example, in certain embodiments, a FZD-binding agentor antibody that binds to more than one FZD, binds to those FZDs with aK_(D) of about 100 nM or less, about 20 nM or less, or about 10 nM orless. In certain embodiments, the FZD-binding agent or antibody binds toeach of one or more (e.g., 1, 2, 3, 4, or 5) of the following FZDs witha dissociation constant of about 40 nM or less: FZD1, FZD2, FZD5, FZD7,and FZD8. In certain embodiments, the FZD-binding agent or antibodybinds to each of one or more of the following FZDs with a dissociationconstant of about 10 nM or less: FZD1, FZD2, FZD5, FZD7, and FZD8. Incertain embodiments, the FZD-binding agent or antibody binds to each ofthe following FZDs with a dissociation constant of about 1 nM or less:FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, thedissociation constant of the agent or antibody to a particular FZD isthe dissociation constant determined using an FZD-Fc fusion proteincomprising the FZD extracellular domain or Fri domain immobilized on aBiacore chip.

In certain embodiments, a FZD-binding agent (e.g., anti-FZD antibody)that is useful in the methods of the invention is an antagonist of atleast one human frizzled receptor (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 FZDs) bound by the agent. In certain embodiments, the agent inhibitsat least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 75%, at least about 90%, or about 100% of oneor more activity of the bound human frizzled receptor.

In certain embodiments, the FZD-binding agent (e.g., anti-FZD antibody)inhibits binding of a ligand to the at least one human frizzledreceptor. In certain embodiments, the ligand is a human Wnt protein.Nineteen human Wnt proteins have been identified: Wnt1, Wnt2, Wnt2B/13,Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A(previously Wnt14), Wnt9B (previously Wnt15), Wnt10A, Wnt10B, Wnt11, andWnt16. In certain embodiments, the agent inhibits binding of Wnt3A toFZD8. In certain embodiments, the inhibition of binding of a particularligand to a particular human frizzled protein provided by theFZD-binding agent is at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, at least about 90%, or at least about95%. In certain embodiments, an agent that inhibits binding of a ligandsuch as a Wnt to a FZD, further inhibits Wnt signaling (e.g., inhibitscanonical Wnt signaling).

In certain embodiments, the FZD-binding agent (e.g., anti-FZD antibody)inhibits Wnt signaling It is understood that a FZD-binding agent thatinhibits Wnt signaling may, in certain embodiments, inhibit signaling byone or more Wnts, but not necessarily by all Wnts. In certainalternative embodiments, signaling by all human Wnts may be inhibited.In certain embodiments, signaling by one or more Wnts selected from thegroup consisting of Wnt1, Wnt2, Wnt2B/13, Wnt3, Wnt3A, Wnt4, Wnt5A,Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A (previously Wnt14), Wnt9B(previously Wnt15), Wnt10A, Wnt10B, Wnt11, and Wnt16 is inhibited. Incertain embodiments, the Wnt signaling that is inhibited is signaling byWnt1, Wnt2, Wnt3, Wnt3A, Wnt7a, Wnt7b, and/or Wnt10B. In certainembodiments, the agent inhibits signaling by (at least) Wnt1, Wnt3A,Wnt7b, and Wnt10B. In particular embodiments, the agent inhibitssignaling by (at least) Wnt3A. In certain embodiments, the inhibition ofsignaling by a Wnt provided by the FZD-binding agent is a reduction inthe level of signaling by the Wnt of least about 10%, at least about25%, at least about 50%, at least about 75%, at least about 90%, or atleast about 95%. In certain embodiments, the Wnt signaling that isinhibited is canonical Wnt signaling.

In vivo and in vitro assays for determining whether a FZD-binding agent(or candidate FZD-binding agent) inhibits Wnt signaling are known in theart. See, e.g., U.S. Pat. Appl. Pub. No. 2012/0027778, which isincorporated by reference herein in its entirety.

In certain embodiments, the FZD-binding agents (e.g., anti-FZDantibodies) useful in the methods of the invention have one or more ofthe following effects: inhibit proliferation of neuroendocrine tumorcells, reduce the tumorigenicity of a neuroendocrine tumor by reducingthe frequency of cancer stem cells in the neuroendocrine tumor, inhibitneuroendocrine tumor growth, increase survival, trigger cell death ofneuroendocrine tumor cells, differentiate tumorigenic neuroendocrinetumor cells to a non-tumorigenic state, or prevent metastasis of tumorcells.

In certain embodiments, the FZD-binding agents useful in the methods ofthe invention are capable of inhibiting neuroendocrine tumor growth. Incertain embodiments, the FZD-binding agents are capable of inhibitingneuroendocrine tumor growth in vivo (e.g., in a xenograft mouse modeland/or in a human having cancer).

In certain embodiments, the FZD-binding agents useful in the methods ofthe invention are capable of reducing the tumorigenicity of aneuroendocrine tumor. In certain embodiments, the agent or antibody iscapable of reducing the tumorigenicity of a neuroendocrine tumorcomprising cancer stem cells in an animal model, such as a mousexenograft model. In certain embodiments, the number or frequency ofcancer stem cells in a tumor is reduced by at least about two-fold,about three-fold, about five-fold, about ten-fold, about 50-fold, about100-fold, or about 1000-fold. In certain embodiments, the reduction inthe number or frequency of cancer stem cells is determined by limitingdilution assay using an animal model. An example of a limiting dilutionassay used to test the efficacy of an anti-FZD antibody is provided inExample 8 of US 2012/0027778, which is incorporated by reference hereinin its entirety. Additional examples and guidance regarding the use oflimiting dilution assays to determine a reduction in the number orfrequency of cancer stem cells in a tumor can be found, e.g., inInternational Publication Number WO 2008/042236, U.S. Patent ApplicationPublication No. 2008/0064049, and U.S. Patent Application PublicationNo. 2008/0178305, each of which is incorporated by reference herein inits entirety.

In certain embodiments, the FZD-binding agent (e.g., antibody) useful inthe methods of the invention is a polypeptide. In certain embodiments,the agent or polypeptide is an antibody. In certain embodiments, theantibody is an IgG1 antibody or an IgG2 antibody. In certainembodiments, the antibody is a monoclonal antibody. In certainembodiments, the antibody is a human antibody or a humanized antibody.In certain embodiments, the antibody is an antibody fragment.

In certain embodiments, an anti-FZD antibody for the methods of theinvention comprise one, two, three, four, five and/or six of the CDRs ofthe 18R5, 18R8 and/or 44R24 human antibodies (see Table 1 below) with upto four (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutionsper CDR. In certain embodiments, the heavy chain CDR(s) are containedwithin a heavy chain variable region and/or the light chain CDR(s) arecontained within a light chain variable region.

TABLE 1 CDRs of 18R8, 18R5, and 44R24 human antibodies Heavy Chain Ab(s)CDR1 CDR2 CDR3 18R8 GFTFSHYTLS VISGDGSYTYYADSVKG NFIKYVFAN(SEQ ID NO: 31) (SEQ ID NO: 32) (SEQ ID NO: 33) 18R5 GFTFSHYTLSVISGDGSYTYYADSVKG NFIKYVFAN (SEQ ID NO: 31) (SEQ ID NO: 32)(SEQ ID NO: 33) 44R24 GFTFSSYYIT TISYSSSNTYYADSVKG SIVFDY(SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48) Light Chain Ab(s) CDR1CDR2 CDR3 18R8 SGDKLGKKYAS EKDNRPSG SSFAGNSLE (SEQ ID NO: 41)(SEQ ID NO: 42) (SEQ ID NO: 43) 18R5 SGDNIGSFYVH DKSNRPSG QSYANTLSL(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) 44R24 SGDALGNRYVY SGGSWDTRPYPKY (SEQ ID NO: 49) (SEQ ID NO: 50) (SEQ ID NO: 51) *Sitedirected change introduced to CDR1 to remove N-linked glycosylation siteis underlined.

In one embodiment, an anti-FZD antibody that is useful in the methods ofthe invention comprises a heavy chain variable region comprising: (a) aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:31), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavychain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:32), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) aheavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:33), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In certainembodiments, the anti-FZD antibody further comprises a light chainvariable region comprising: (a) a light chain CDR1 comprisingSGDKLGKKYAS (SEQ ID NO:41), or a variant of thereof comprising 1, 2, 3,or 4 amino acid substitutions; (b) a light chain CDR2 comprisingEKDNRPSG (SEQ ID NO:42), or a variant of thereof comprising 1, 2, 3, or4 amino acid substitutions; and/or (c) a light chain CDR3 comprisingSSFAGNSLE (SEQ ID NO:43), or a variant of thereof comprising 1, 2, 3, or4 amino acid substitutions. In certain embodiments, the amino acidsubstitutions are conservative substitutions. In a further embodiment,an anti-FZD antibody that is useful in the methods of the inventioncomprises (a) a heavy chain variable region comprising a heavy chainCDR1 comprising GFTFSHYTLS (SEQ ID NO:31), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:32), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:33); and/or (b) a light chain variable regioncomprising a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:41), alight chain CDR2 comprising EKDNRPSG (SEQ ID NO:42), and/or a lightchain CDR3 comprising SSFAGNSLE (SEQ ID NO:43).

In one embodiment, an anti-FZD antibody that is useful in the methods ofthe invention comprises a heavy chain variable region comprising: (a) aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:31), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavychain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:32), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) aheavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:33), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In certainembodiments, the anti-FZD antibody further comprises a light chainvariable region comprising: (a) a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:34), or a variant of thereof comprising 1, 2, 3,or 4 amino acid substitutions; (b) a light chain CDR2 comprisingDKSNRPSG (SEQ ID NO:35), or a variant of thereof comprising 1, 2, 3, or4 amino acid substitutions; and/or (c) a light chain CDR3 comprisingQSYANTLSL (SEQ ID NO:36), or a variant of thereof comprising 1, 2, 3, or4 amino acid substitutions. In certain embodiments, the amino acidsubstitutions are conservative substitutions. In a further embodiment,an anti-FZD antibody that is useful in the methods of the inventioncomprises (a) a heavy chain variable region comprising a heavy chainCDR1 comprising GFTFSHYTLS (SEQ ID NO:31), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:32), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:33); and/or (b) light chain variable regioncomprising a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:34), alight chain CDR2 comprising DKSNRPSG (SEQ ID NO:35), and a light chainCDR3 comprising QSYANTLSL (SEQ ID NO:36).

In one embodiment, an anti-FZD antibody that is useful in the methods ofthe invention comprises a heavy chain variable region comprising: (a) aheavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO:46), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions;(b) a heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO:47), or avariant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; and/or (c) a heavy chain CDR3 comprising SIVFDY (SEQ IDNO:48), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions. In certain embodiments, the anti-FZD antibodyfurther comprises a light chain variable region comprising: (a) a lightchain CDR1 comprising SGDALGNRYVY (SEQ ID NO:49), or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; (b) alight chain CDR2 comprising SG (SEQ ID NO:50), or a variant thereofcomprising 1, 2, 3, or 4 conservative amino acid substitutions; and (c)a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO:51), or a variantthereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.In certain embodiments, the antibody comprises: (a) a heavy chain CDR1comprising GFTFSSYYIT (SEQ ID NO:46), a heavy chain CDR2 comprisingTISYSSSNTYYADSVKG (SEQ ID NO:47), and a heavy chain CDR3 comprisingSIVFDY (SEQ ID NO:48); and/or (b) a light chain CDR1 comprisingSGDALGNRYVY (SEQ ID NO:49), a light chain CDR2 comprising SG (SEQ IDNO:50), and a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO:51).

In certain embodiments, an anti-FZD antibody useful for the methods ofthe invention comprise: (a) a heavy chain variable region having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, or at least about 99% sequence identity to SEQID NO:37 or SEQ ID NO:52; and/or (b) a light chain variable regionhaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 97%, or at least about 99% sequenceidentity to SEQ ID NO:44, SEQ ID NO:38 or SEQ ID NO:53. In certainembodiments, an anti-FZD antibody useful for the methods of theinvention comprise: (a) a heavy chain variable region having at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 97%, or at least about 99% sequence identity to SEQ IDNO:37; and (b) a light chain variable region having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 97%, or at least about 99% sequence identity to SEQ ID NO:38. Incertain embodiments, the anti-FZD antibody useful for the methods of theinvention comprises (a) a heavy chain variable region having the aminoacid sequence of SEQ ID NO:37 or SEQ ID NO:52; and/or (b) a light chainvariable region having the amino acid sequence of SEQ ID NO:44, SEQ IDNO:38 or SEQ ID NO:53. In certain embodiments, the anti-FZD antibodycomprises (a) a heavy chain variable region having the amino acidsequence of SEQ ID NO:37; and/or (b) a light chain variable regionhaving the amino acid sequence of SEQ ID NO:44. In certain embodiments,the anti-FZD antibody comprises (a) a heavy chain variable region havingthe amino acid sequence of SEQ ID NO:37; and/or (b) a light chainvariable region having the amino acid sequence of SEQ ID NO:38. Incertain embodiments, the anti-FZD antibody comprises (a) a heavy chainvariable region having the amino acid sequence of SEQ ID NO:52; and/or(b) a light chain variable region having the amino acid sequence of SEQID NO:53.

TABLE 2 VH and VL of selected human anti-FZD antibodies Heavy ChainVariable Region Light Chain Variable Region Ab(s) (VH) amino acidsequence (VL) amino acid sequence 18R8 SEQ ID NO: 37 SEQ ID NO: 44 18R5SEQ ID NO: 37 SEQ ID NO: 38 44R24 SEQ ID NO: 52 SEQ ID NO: 53

In certain embodiments, an anti-FZD antibody useful for the methods ofthe invention comprises (a) a heavy chain of SEQ ID NO:39 and lightchain of SEQ ID NO:45; or (b) a heavy chain of SEQ ID NO:39 and lightchain of SEQ ID NO:40.

TABLE 3 The heavy chain and light chain of selected human anti-FZDantibodies Heavy Chain Variable Region Light Chain Variable Region Ab(s)(VH) amino acid sequence (VL) amino acid sequence 18R8 SEQ ID NO: 39 SEQID NO: 45 18R5 SEQ ID NO: 39 SEQ ID NO: 40

In certain embodiments, the FZD-binding agent useful in the methods ofthe invention comprises, consists essentially of, or consists of ananti-FZD antibody selected from the group consisting of 18R8, 18R5, and44R24 IgG antibodies.

In certain embodiments, the FZD-binding agent useful in the methods ofthe invention comprises the heavy chains and light chains of the 18R8IgG2 antibody (with or without the leader sequence). In certainembodiments, the FZD-binding agent is the 18R8 IgG2 antibody. DNAencoding the heavy chains and light chains of the 18R8 IgG2 antibody wasdeposited with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., USA, under the conditions of theBudapest Treaty on Sep. 29, 2008, and assigned ATCC deposit designationnumber PTA-9540. In certain embodiments, the FZD-binding agent useful inthe methods of the invention comprises the heavy chains and light chainsof the 18R5 IgG2 antibody (with or without the leader sequence). Incertain embodiments, the FZD-binding agent is the 18R5 IgG2 antibody.The 18R5 IgG2 antibody is also referred to herein as OMP-18R5. DNAencoding the heavy chains and light chains of the 18R5 IgG2 antibody wasdeposited with the ATCC, under the conditions of the Budapest Treaty onSep. 29, 2008, and assigned ATCC deposit designation number PTA-9541.Additional information regarding the OMP-18R5 antibody can be found, forexample, in U.S. Pat. No. 7,982,013, which is incorporated by referenceherein in its entirety. In U.S. Pat. No. 7,982,013, the OMP-18R5antibody is generally referred to as “18R5” or the “18R5 IgG2 antibody.”

In certain embodiments, the FZD-binding agent useful in the methods ofthe invention is an IgG antibody encoded by the plasmid deposited withthe ATCC on Aug. 26, 2009, and assigned deposit designation numberPTA-10307, PTA-10309, or PTA-10311.

In certain embodiments, the FZD-binding agent useful in the methods ofthe invention is an agent that competes for specific binding to FZD1,FZD2, FZD5, FZD7, and/or FZD8 with an antibody encoded by the plasmidhaving ATCC deposit designation number PTA-9540, PTA-9541, PTA-10307, orPTA-10309 (e.g., in a competitive binding assay). In certain alternativeembodiments, the FZD-binding agent is an agent that competes forspecific binding to FZD5 and/or FZD8 with an antibody encoded by theplasmid having ATCC deposit designation number PTA-10311.

In certain embodiments, the FZD-binding agent (e.g., antibody) useful inthe methods of the invention binds to the same epitope as or binds to anepitope that overlaps with the epitope of the 18R5, 18R8, or 44R24antibody.

In certain embodiments, the FZD-binding agent FZD-binding agent (e.g.,antibody) useful in the methods of the invention competes for specificbinding to a human frizzled receptor with the 18R5, 18R8, or 44R24antibody.

Further examples of FZD-binding agents useful in the methods of theinvention are disclosed in U.S. Pat. Appl. Pub. No. 2012/0027778, whichis incorporated by reference herein in its entirety.

In certain embodiments, the FZD-binding agent useful in the methods ofthe invention has a circulating half-life in mice, cynomolgous monkeys,or humans of at least about 10 hours, at least about 24 hours, at leastabout 3 days, at least about 1 week, or at least about 2 weeks. Incertain embodiments, the FZD-binding agent is an IgG (e.g., IgG1 orIgG2) antibody that has a circulating half-life in mice, cynomolgousmonkeys, or humans of at least about 10 hours, at least about 24 hours,at least about 3 days, at least about 1 week, or at least about 2 weeks.Methods of increasing the half-life of agents such as polypeptides andantibodies are known in the art. For example, known methods ofincreasing the circulating half-life of IgG antibodies include theintroduction of mutations in the Fc region which increase thepH-dependent binding of the antibody to the neonatal Fc receptor (FcRn)at pH 6.0 (see, e.g., U.S. Pat. Pub. Nos. 2005/0276799, 2007/0148164,and 2007/0122403). Known methods of increasing the circulating half-lifeof antibody fragments lacking the Fc region include such techniques asPEGylation.

In certain embodiments, an anti-FZD antibody useful for the methods ofthe invention is a bispecific antibody that specifically recognizes ahuman frizzled receptor. Bispecific antibodies are antibodies that arecapable of specifically recognizing and binding at least two differentepitopes. In one embodiment, the bispecific anti-FZD antibodyspecifically recognizes different epitopes within the same humanfrizzled receptor. In another embodiment, the bispecific anti-FZDantibody specifically recognizes different epitopes within a humanfrizzled receptor or on different human frizzled receptors.

Alternatively, in certain alternative embodiments, an anti-FZD antibodyuseful for the methods of the invention is not a bispecific antibody.

In certain embodiments, an anti-FZD antibody useful for the methods ofthe invention is monospecific. For example, in certain embodiments, eachof the one or more antigen-binding sites that an antibody contains iscapable of binding (or binds) the same one or more human FZD receptors(e.g., FZD1, FZD2, FZD5, FZD7, or FZD8, or a homologous epitope on somecombination of the FZDs). In certain embodiments, an antigen-bindingsite of the monospecific anti-FZD antibody is capable of binding (orbinds) one, two, three, four, or five (or more) human frizzledreceptors.

In certain embodiments, the FZD-binding agent useful for the methods ofthe invention is a polypeptide that is not an antibody. A variety ofmethods for identifying and producing non-antibody polypeptides thatbind with high affinity to a protein target are known in the art. See,e.g., Skerra, Curr. Opin. Biotechnol., 18:295-304 (2007), Hosse et al.,Protein Science, 15:14-27 (2006), Gill et al., Curr. Opin. Biotechnol.,17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBSJ., 275:2677-83 (2008), each of which is incorporated by referenceherein in its entirety.

In certain embodiments, the FZD-binding agent useful for the methods ofthe invention comprises a protein scaffold of a type selected from thegroup consisting of protein A, a lipocalin, a fribronectin domain, anankyrin consensus repeat domain, and thioredoxin.

In certain embodiments, the FZD-binding agent useful for the methods ofthe invention has been naturally or unnaturally modified. By way ofnon-limiting example, the polypeptide may be labeled. In certainembodiments, the polypeptide is glycosylated, pegylated, phosphorylated,or acetylated, amidated. In certain embodiments, the modificationsincrease stability and/or the in vivo half-life of the polypeptide. Incertain embodiments, the polypeptides are cyclic. In certain furtherembodiments, the polypeptides comprise one or more N-methyl amino acids.

In certain embodiments, the FZD-binding agent useful for the methods ofthe invention is (or comprises) a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:54-72, or (b)an amino acid sequence having at least about 80%, at least about 85%, atleast about 88%, or at least about 90% amino acid sequence identity to asequence selected from the group consisting of SEQ ID NOs:54-67 or69-72. In certain embodiments, the polypeptides comprise, consistessentially of, or consist of a cyclic peptide selected from the groupconsisting of SEQ ID NOs:54-72. In certain embodiments, the amino acidsequence is SEQ ID NO:64. In certain alternative embodiments, the aminoacid sequence is SEQ ID NO:68.

In certain embodiments, the FZD-binding polypeptide useful for themethods of the invention is less than about 500 amino acids in length,less than about 200 amino acids in length, less than about 100 aminoacids in length, less than about 50 amino acids in length, less thanabout 20 amino acids amino acids in length, or less than about 15 aminoacids in length. In certain embodiments, the FZD-binding polypeptide isat least about 3, at least about 5, or at least about 7 amino acids inlength. Accordingly, in certain embodiments the polypeptide is betweenabout 5 and about 20 amino acids in length. In some embodiments, thepolypeptide is between about 7 and about 15 amino acids in length.

4. Soluble Receptors

An additional aspect of the methods of the invention is the use of Wntantagonist soluble receptors in the treatment of neuroendocrine tumors.In certain embodiments, the soluble receptor useful in the methods ofthe invention comprises the extracellular domain of a FZD receptor. Insome embodiments, the soluble receptor useful in the methods of theinvention comprises a Fri domain of a FZD receptor. In certainembodiments, the FZD receptor is a human FZD receptor. In certainembodiments, the human FZD receptor is FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, or FZD10. In certain embodiments, the FZDreceptor is FZD8. In certain embodiments, the Wnt antagonist used in themethods described herein comprises a human FZD8 Fri domain and a humanFc region.

In some alternative embodiments, the soluble receptor useful in themethods of the invention comprises a portion of a SFRP. In someembodiments, the soluble receptor useful in the methods of the inventioncomprises a Fri domain of a SFRP. In certain embodiments, the SFRP is ahuman SFRP. In some embodiments, the human SFRP is SFRP1, SFRP2, SFRP3,SFRP4, or SFRP5. The minimal, core Fri domain sequences for each of thehuman SFRPs (SFRP1-5) are provided as SEQ ID NOs:83-87.

In other alternative embodiments, the soluble receptor useful in themethods of the invention comprises the extracellular domain of a Rorprotein. In some embodiments, the soluble receptor useful in the methodsof the invention comprises a Fri domain of a Ror protein. In certainembodiments, the Ror is a human Ror. In some embodiments, the human Roris Ror1 or Ror2. The minimal, core Fri domain sequences of human Ror1and Ror2 are provided as SEQ ID NO:88 and SEQ ID NO:89.

In certain embodiments, the soluble receptors (e.g., FZD8 Fri.Fc) thatare useful in the methods of the invention specifically bind one, two,three, four, five, six, seven, eight, nine, ten, or more Wnt proteins.By way of non-limiting example, the Wnt-binding agent may bind Wnt1,Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and/orWnt10b. In certain embodiments, the Wnt-binding agent binds Wnt1, Wnt2,Wnt3, Wnt3a, and Wnt7b. In certain embodiments, the soluble receptor isa Wnt antagonist. In certain embodiments, the soluble receptor inhibitsWnt-signaling. In some embodiments, the soluble receptor inhibitscanonical Wnt signaling.

Nonlimiting examples of soluble FZD receptors useful in the methods ofthe invention can be found in U.S. Pat. No. 7,723,477, which isincorporated by reference herein in its entirety. Additional solublereceptors (e.g., soluble FZD receptors) are disclosed in US2011/0305695, which is incorporated by reference herein in its entirety.

In certain embodiments, a soluble receptor useful in the methods of theinvention comprises a Fri domain of a human FZD receptor, or a fragmentor variant of the Fri domain that binds one or more human Wnt proteins.In certain embodiments, the human FZD receptor is FZD4. In certainalternative embodiments, the human FZD receptor is FZD5. In certainadditional alternative embodiments, the human FZD receptor is FZD8. Incertain embodiments, the FZD is FZD4 and the soluble receptor comprisesSEQ ID NO:76 or comprises approximately amino acids 40 to 170 of SEQ IDNO:90. In certain embodiments, the FZD is FZD5 and the soluble receptorcomprises SEQ ID NO:77 or comprises approximately amino acids 27-157 ofSEQ ID NO:91. In certain embodiments, the FZD is FZD8 and the solublereceptor comprises SEQ ID NO:80 or comprises approximately amino acids28-158 of SEQ ID NO:92.

In certain embodiments, the soluble receptor useful in the methods ofthe invention comprises a minimal Fri domain sequence selected from thegroup consisting of SEQ ID NOs:73-89. In certain embodiments, thesoluble receptor useful in the methods of the invention comprises avariant of any one of the aforementioned Fri domain sequences thatcomprises one or more (e.g., one, two, three, four, five, six, seven,eight, nine, ten, etc.) conservative substitutions and is capable ofbinding Wnt(s).

In certain embodiments, the soluble receptor useful in the methods ofthe invention, such as a soluble receptor comprising a minimum Fridomain of a human FZD receptor, further comprises a human Fc region(e.g., a human IgG1 Fc region). Soluble receptors comprising the Fridomain of a FZD receptor and human IgG1 Fc are referred to herein as“FZD Fri.Fc” (e.g. FZD8 Fri.Fc). The Fc region can be obtained from anyof the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. In someembodiments, the Fc region is a wild-type Fc region. In someembodiments, the Fc region is a mutated Fc region. In some embodiments,the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids, (e.g., in the hinge domain). In someembodiments, an amino acid in the hinge domain is changed to hinderundesirable disulfide bond formation. In some embodiments, a cysteine isreplaced with a serine to hinder undesirable disulfide bond formation.In certain embodiments, the Fc region comprises or consists of SEQ IDNO:93, SEQ ID NO:94, or SEQ ID NO:95.

In certain embodiments, a soluble receptor useful in the methods of theinvention is a fusion protein comprising at least a minimum Fri domain(e.g., a minimum Fri domain of a FZD receptor) and an Fc region. As usedherein, a “fusion protein” is a hybrid protein expressed by a nucleicacid molecule comprising nucleotide sequences of at least two genes. Insome embodiments, the C-terminus of the first polypeptide is linked tothe N-terminus of the immunoglobulin Fc region. In some embodiments, thefirst polypeptide (e.g., a FZD Fri domain) is directly linked to the Fcregion (i.e. without an intervening peptide linker). In someembodiments, the first polypeptide is linked to the Fc region via apeptide linker.

As used herein, the term “linker” refers to a linker inserted between afirst polypeptide (e.g., a FZD component) and a second polypeptide(e.g., an Fc region). In some embodiments, the linker is a peptidelinker. Linkers should not adversely affect the expression, secretion,or bioactivity of the polypeptide. Linkers should not be antigenic andshould not elicit an immune response. Suitable linkers are known tothose of skill in the art and often include mixtures of glycine andserine residues and often include amino acids that are stericallyunhindered. Other amino acids that can be incorporated into usefullinkers include threonine and alanine residues. Linkers can range inlength, for example from 1-50 amino acids in length, 1-22 amino acids inlength, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3amino acids in length. Linkers may include, but are not limited to,SerGly, GGSG, GSGS, GGGS, S(GGS). where n is 1-7, GRA, poly(Gly),poly(Ala), ESGGGGVT (SEQ ID NO:96), LESGGGGVT (SEQ ID NO:97), GRAQVT(SEQ ID NO:98), WRAQVT (SEQ ID NO:99), and ARGRAQVT (SEQ ID NO:100). Asused herein, a linker is an intervening peptide sequence that does notinclude amino acid residues from either the C-terminus of the firstpolypeptide (e.g., a FZD Fri domain) or the N-terminus of the secondpolypeptide (e.g., the Fc region).

In certain embodiments, soluble receptors useful for the methods of theinvention contain a signal sequence that directs the transport of theproteins. Signal sequences (also referred to as signal peptides orleader sequences) are located at the N-terminus of nascent polypeptides.They target the polypeptide to the endoplasmic reticulum and theproteins are sorted to their destinations, for example, to the innerspace of an organelle, to an interior membrane, to the cell's outermembrane, or to the cell exterior via secretion. Most signal sequencesare cleaved from the protein by a signal peptidase after the proteinsare transported to the endoplasmic reticulum. The cleavage of the signalsequence from the polypeptide usually occurs at a specific site in theamino acid sequence and is dependent upon amino acid residues within thesignal sequence. Although there is usually one specific cleavage site,more than one cleavage site may be recognized and/or used by a signalpeptidase resulting in a non-homogenous N-terminus of the polypeptide.For example, the use of different cleavage sites within a signalsequence can result in a polypeptide expressed with different N-terminalamino acids. Accordingly, in some embodiments, the soluble receptorsuseful for the methods of the invention may comprise a mixture ofpolypeptides with different N-termini. In some embodiments, theN-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In someembodiments, the soluble receptor polypeptide is substantiallyhomogeneous, i.e., the polypeptides have the same N-terminus. In someembodiments, the signal sequence of the polypeptide comprises amino acidsubstitutions and/or deletions that allow one cleavage site to bedominant, thereby resulting in a substantially homogeneous polypeptidewith one N-terminus. In some embodiments, the signal sequence of thepolypeptide comprises or consists of a sequence selected from the grouplisted in Table 3. In some embodiments, the signal sequence is SEQ IDNO:101. In some embodiments, the signal sequence is SEQ ID NO:104. Insome embodiments, the signal sequence is SEQ ID NO:106.

TABLE 3 Signal sequences. MEWGYLLEVTSLLAALALLQRSSGAAA SEQ ID NO: 101MEWGYLLEVTSLLAALALLQRSSGALA SEQ ID NO: 102 MEWGYLLEVTSLLAALALLQRSSGVLASEQ ID NO: 103 MEWGYLLEVTSLLAALLLLQRSPIVHA SEQ ID NO: 104MEWGYLLEVTSLLAALFLLQRSPIVHA SEQ ID NO: 105 MEWGYLLEVTSLLAALLLLQRSPFVHASEQ ID NO: 106 MEWGYLLEVTSLLAALLLLQRSPIIYA SEQ ID NO: 107MEWGYLLEVTSLLAALLLLQRSPIAHA SEQ ID NO: 108

In certain embodiments, a soluble receptor useful in the methods of theinvention comprises a first polypeptide comprising a FZD domaincomponent and an Fc region. In some embodiments, the FZD domaincomponent is from FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9,or FZD10. In some embodiments, the Fc region is from an IgG1immunoglobulin. In some embodiments, the soluble receptor comprises: (a)a first polypeptide consisting essentially of amino acids selected fromthe group consisting of: X1 to Y1 of SEQ ID NO:11, X2 to Y2 of SEQ IDNO:12, X3 to Y3 of SEQ ID NO:13, X4 to Y4 of SEQ ID NO:14, X5 to Y5 ofSEQ ID NO:15, X6 to Y6 of SEQ ID NO:16, X7 to Y7 of SEQ ID NO:17, X8 toY8 of SEQ ID NO:18, X9 to Y9 of SEQ ID NO:19, and X10 to Y10 of SEQ IDNO:20; and

(b) a second polypeptide consisting essentially of amino acids A to B ofSEQ ID NO:95; wherein X1=amino acid 69, 70, 71,72, 73, 74, 75, or 76

-   -   Y1=amino acid 236, 237, 238, 239, 240, 241, 242, or 243    -   X2=amino acid 22, 23, 24, 25, 26, 27 or 28    -   Y2=amino acid 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,        168, 169, 170, 171 or 172    -   X3=amino acid 18, 19, 20, 21, 22, 23, 24, or 25    -   Y3=amino acid 141, 142, 143, 144, 145, 146, 147, 148, or 149    -   X4=amino acid 38, 39, 40, 41, or 42    -   Y4=amino acid 168, 169, 170, 171, 172, 173, 174, 175 or 176    -   X5=amino acid 25, 26, 27, 28 or 29    -   Y5=amino acid 155, 156, 157, 158, 159, 160, 161, 162, 163, or        164    -   X6=amino acid 19, 20, 21, 22, 23, or 24    -   Y6=amino acid 144, 145, 146, 147, 148, 149, 150, 151 or 152    -   X7=amino acid 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or        34    -   Y7=amino acid 178, 179, 180, 181, 182, 183, 184, 185, or 186    -   X8=amino acid 25, 26, 27, 28, 29, 30, or 31    -   Y8=amino acid 156, 157, 158, 159, 160, 161, 162, 163, or 164    -   X9=amino acid 21, 22, 23, or 24    -   Y9=amino acid 137, 138, 139, 140, 141, 142, 143, 144, 145, or        146    -   X10=amino acid 20, 21, 22, 23, 24, or 25    -   Y10=amino acid 152, 153, 154, 155, 156, 157, 158, 159, or 160    -   A=amino acid 1, 2, 3, 4, 5, or 6    -   B=amino acid 231 or 232.

In some embodiments, the first polypeptide is directly linked to thesecond polypeptide. In some embodiments, the first polypeptide is linkedto the second polypeptide via a peptide linker. In some embodiments, thefirst polypeptide is linked to the second polypeptide via the peptidelinker GRA. A polypeptide (e.g., a first or second polypeptide) that“consists essentially of”60 certain amino acids or is “consistingessentially of” certain amino acids may, in some embodiments, includeone or more (e.g., one, two, three, four or more) additional amino acidsat one or both ends, so long as the additional amino acids do notmaterially affect the function of the Wnt-binding agent.

In certain embodiments, a soluble receptor useful in the methods of theinvention comprises:

(a) a first polypeptide consisting essentially of amino acids X to Y ofSEQ ID NO:18; and (b) a second polypeptide consisting essentially ofamino acids A to B of SEQ ID NO:95; wherein the first polypeptide isdirectly linked to the second polypeptide; and wherein

X=amino acid 25, 26, 27, 28, 29, 30, or 31

Y=amino acid 156, 157, 158, 159, 160, 161, 162, 163, or 164

A=amino acid 1, 2, 3, 4, 5, or 6

B=amino acid 231 or 232.

In some embodiments, the first polypeptide consists essentially of aminoacids 25-158 of SEQ ID NO:18. In other embodiments, the firstpolypeptide consists of amino acids 25-158 of SEQ ID NO:18. In someembodiments, the first polypeptide consists essentially of amino acids28-158 of SEQ ID NO:18. In other embodiments, the first polypeptideconsists of amino acids 28-158 of SEQ ID NO:18. In some embodiments, thefirst polypeptide consists of amino acids 31-158 of SEQ ID NO:18. Insome embodiments, the second polypeptide consists of amino acids 1-232of SEQ ID NO:95. In some embodiments, the second polypeptide consists ofamino acids 3-232 of SEQ ID NO:95. In some embodiments, the secondpolypeptide consists of amino acids 6-232 of SEQ ID NO:95. In someembodiments, the first polypeptide is SEQ ID NO:28 and the secondpolypeptide is SEQ ID NO:95. In some embodiments, the first polypeptideis SEQ ID NO:28 and the second polypeptide is SEQ ID NO:94. In someembodiments, the first polypeptide is SEQ ID NO:28 and the secondpolypeptide is SEQ ID NO:93.

In some embodiments, the soluble receptor useful in the methods of theinvention comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:109-121. In certain alternative embodiments, thesoluble receptor comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:109-121, comprising one or more (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, etc.)conservative substitutions. In certain embodiments, soluble receptorcomprises a sequence having at least about 90%, about 95%, or about 98%sequence identity with an amino acid sequence selected from the groupconsisting of SEQ ID NO:109-121. In certain embodiments, the variantsoluble receptor maintains its ability to bind one or more human Wnts.

In certain embodiments, the soluble receptor useful in the methods ofthe invention comprises the sequence of SEQ ID NO:109. In certainembodiments, the soluble receptor comprises the sequence of SEQ IDNO:115. In some embodiments, the soluble receptor consists of ahomodimer formed by polypeptides consisting of SEQ ID NO:115. In certainembodiments, the soluble receptor comprises the sequence of SEQ IDNO:117. In some embodiments, the soluble receptor consists of ahomodimer formed by polypeptides consisting of SEQ ID NO:117.

In some embodiments, the soluble receptors (e.g., FZD8 Fri.Fc) useful inthe methods of the invention inhibit the growth of a neuroendocrinetumor or tumor cells. In some embodiments, the soluble receptors induceneuroendocrine tumor cells to differentiate. In some embodiments, thesoluble receptors induce the expression of differentiation markers on aneuroendocrine tumor or tumor cell. In certain embodiments, the solublereceptors reduce the frequency of cancer stem cells in a neuroendocrinetumor. In certain embodiments, the soluble receptors inhibit the growthof a Wnt-dependent neuroendocrine tumor. In some embodiments, a solublereceptor comprising SEQ ID NO:115 inhibits neuroendocrine tumor growthto a greater extent than a soluble receptor comprising SEQ ID NO:109. Insome embodiments, a soluble receptor comprising SEQ ID NO:117 inhibitsneuroendocrine tumor growth to a greater extent than a soluble receptorcomprising SEQ ID NO:109. In some embodiments, a soluble receptorinhibits tumor growth to a greater extent than a soluble receptorcomprising a FZD domain component, an Fc domain and a linker componentconnecting the FZD domain component and the Fc domain. In someembodiments, the linker component is an intervening peptide linker.

In certain embodiments, the soluble receptor useful in the methods ofthe invention (before signal sequence cleavage) comprises SEQ ID NO:115and a signal sequence selected from the group consisting of SEQ ID NO:104-108. In some embodiments, the soluble receptor (before signalsequence cleavage) comprises SEQ ID NO:117 and a signal sequenceselected from the group consisting of SEQ ID NO: 104-108. In someembodiments, the soluble receptor comprises SEQ ID NO:105 and SEQ IDNO:115. In some embodiments, the soluble receptor comprises SEQ IDNO:105 and SEQ ID NO:117. In some embodiments, the soluble receptorcomprises SEQ ID NO:106 and SEQ ID NO:115. In some embodiments, thesoluble receptor comprises SEQ ID NO:106 and SEQ ID NO:117. In someembodiments, the soluble receptor comprises SEQ ID NO:133.

In some embodiments, the soluble receptor (e.g., FZD8 Fri.Fc) is asubstantially purified polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, and SEQ ID NO:117. In certain embodiments, thesubstantially purified soluble receptor polypeptide comprises at least80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least99% polypeptide that has an N-terminal sequence of ASA. In certainembodiments, the substantially purified soluble receptor polypeptideconsists of a polypeptide that has an N-terminal sequence of ASA. Insome embodiments, the nascent soluble receptor polypeptide comprises asignal sequence selected from the group consisting of SEQ ID NOs:101-108. In some embodiments, the nascent soluble receptor polypeptidecomprises a signal sequence of SEQ ID NO:106. In some embodiments, thenascent soluble receptor polypeptide comprises a signal sequence thatresults in a substantially homogeneous polypeptide product with oneN-terminal sequence.

In certain embodiments, the soluble FZD receptor polypeptide isOMP-54F28. OMP-54F28 is a homodimer formed by two polypeptide chainsthat each consists of SEQ ID NO:117. Additional information regardingOMP-54F28 can be found in U.S. Pat. Appl. Pub. No. 2011/0305695, whichis incorporated by reference herein in its entirety. OMP-54F28 isgenerally referred to as “54F28” in U.S. Pat. Appl. Pub. No.2011/0305695.

In certain embodiments, a soluble receptor (e.g., FZD8 Fri.Fc) useful inthe methods of the invention comprises an Fc region of animmunoglobulin. In certain embodiments, at least a portion of the Fcregion has been deleted or otherwise altered so as to provide desiredbiochemical or biological characteristics, such as increased cancer celllocalization, increased tumor penetration, reduced serum half-life, orincreased serum half-life, reduced or no ADCC activity, reduced or nocomplement-dependent cytotoxicity (CDC) when compared with a solublereceptor of approximately the same immunogenicity comprising a native orunaltered Fc constant region. Modifications to the Fc region may includeadditions, deletions, or substitutions of one or more amino acids in oneor more domains. Additional soluble receptors (e.g., soluble FZDreceptors) comprising a modified Fc region are disclosed in US2011/0305695, which is incorporated by reference herein in its entirety.

In certain embodiments, the soluble receptors (e.g., FZD8 Fri.Fc) usefulin the methods of the invention bind to at least one Wnt with adissociation constant (K_(D)) of about 1 μM or less, about 100 nM orless, about 40 nM or less, about 20 nM or less, or about 10 nM or less.The soluble receptors can be assayed for specific binding by any methodknown in the art. Such assays are routine and well known in the art(see, e.g., Ausubel et al, eds, 1994, Current Protocols in MolecularBiology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety).

In certain embodiments, the soluble receptor (e.g., FZD8 Fri.Fc) usefulin the methods of the invention (e.g., a FZD8 Fri.Fc) is an antagonistof at least one Wnt (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Wnts) boundby the soluble receptor. In certain embodiments, the soluble receptorinhibits at least about 10%, at least about 20%, at least about 30%, atleast about 50%, at least about 75%, at least about 90%, or about 100%of one or more activity of the bound human Wnt(s). In vivo and in vitroassays for determining whether a soluble receptor inhibits Wnt signalingare known in the art. Suitable methods are disclosed in US 2011/0305695,which is incorporated by reference herein in its entirety.

In certain embodiments, a soluble receptor (e.g., FZD8 Fri.Fc) useful inthe methods of the invention is derivatized with a water solublepolymer. Suitable water soluble polymers include, but are not limitedto, polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), dextran, poly(n-vinyl pyrrolidone)-polyethylene glycol,propropylene glycol homopolymers, prolypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. In certain embodiments, the water solublepolymer is polyethylene glycol (PEG).

In certain embodiments, the soluble receptor (e.g., FZD8 Fri.Fc) usefulin the methods of the invention has a circulating half-life in mice,cynomolgous monkeys, or humans of at least about 5 hours, at least about10 hours, at least about 24 hours, at least about 3 days, at least about1 week, or at least about 2 weeks. In certain embodiments, the solublereceptors have a half-life of at least about 50 hours in a rat whenadministered via the tail vein at a dose ranging from about 2 mg/kg toabout 10 mg/kg. In certain embodiments, the soluble receptor is asoluble FZD receptor that comprises a Fri domain of a human FZD receptor(or a fragment or variant of the Fri domain that binds one or more Wnts)and a human Fc region and has a half-life in vivo (e.g., in a mouse orrat) that is longer than a soluble FZD receptor comprising theextracellular domain of the FZD receptor and a human Fc region.

5. Anti-Wnt Antibodies

A further aspect of the methods of the invention is the use of anti-Wntantibodies in the treatment of neuroendocrine tumors. In certainembodiments, the anti-Wnt antibodies that are useful in the methods ofthe invention specifically bind one or more Wnt polypeptides. In certainembodiments, the antibodies specifically bind two, three, four, five,six, seven, eight, nine, ten or more Wnts. The human Wnt(s) bound by theantibody may be selected from the group consisting of Wnt1, Wnt2, Wnt2b,Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b,Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. In certain embodiments,the one or more (or two or more, three or more, four or more, five ormore, etc.) Wnts bound by the antibody or other antibody comprise Wnt1,Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, andWnt10b. In certain embodiments, the one or more (or two or more, threeor more, four or more, five or more, etc.) Wnts comprise Wnt1, Wnt2,Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, and Wnt10b.

In certain embodiments, an individual antigen-binding site of aWnt-binding antibody useful in the methods of the invention is capableof binding (or binds) the one, two, three, four, or five (or more) humanWnts. In certain embodiments, an individual antigen-binding site of theWnt-binding antibody is capable of specifically binding one, two, three,four, or five human Wnts selected from the group consisting of Wnt1,Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, andWnt10b.

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention binds to the C-terminal cysteine rich domain of a humanWnt. In certain embodiments, the antibody binds to a domain (within theone or more Wnt proteins to which the antibody binds) that is selectedfrom the group consisting of SEQ ID NOs:122-132. In some embodiments,the Wnt-binding antibody binds within SEQ ID NO:122. In someembodiments, the Wnt-binding antibody binds within amino acids 288-370of Wnt1.

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention binds to one or more (for example, two or more, threeor more, or four or more) Wnts with a dissociation constant (K_(D)) ofabout 1 μM or less, about 100 nM or less, about 40 nM or less, about 20nM or less, or about 10 nM or less. For example, in certain embodiments,a Wnt-binding antibody useful in the methods of the invention that bindsto more than one Wnt, binds to those Wnts with a K_(D) of about 100 nMor less, about 20 nM or less, or about 10 nM or less. In certainembodiments, the Wnt-binding antibody binds to each of one or more(e.g., 1, 2, 3, 4, or 5) of the following Wnts with a dissociationconstant of about 40 nM or less: Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a,Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b.

In certain embodiments, the anti-Wnt antibody useful in the methods ofthe invention is an IgG1 antibody or an IgG2 antibody. In certainembodiments, the antibody is a monoclonal antibody. In certainembodiments, the antibody is a human antibody or a humanized antibody.In certain embodiments, the antibody is an antibody fragment.

The antibodies or other antibodies of the present invention can beassayed for specific binding by any method known in the art. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety).

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention is an antagonist of at least one Wnt (i.e., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 Wnts) bound by the antibody. In certainembodiments, the antibody inhibits at least about 10%, at least about20%, at least about 30%, at least about 50%, at least about 75%, atleast about 90%, or about 100% of one or more activity of the boundhuman Wnt(s).

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention inhibits binding of a ligand to the at least one humanWnt. In certain embodiments, the Wnt-binding antibody inhibits bindingof a human Wnt protein to one or more of its ligands. Nineteen human Wntproteins have been identified: Wnt1, Wnt2, Wnt2B/13, Wnt3, Wnt3a, Wnt4,Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a (previouslyWnt14), Wnt9b (previously Wnt15), Wnt10a, Wnt10b, Wnt11, and Wnt16. Tenhuman FZD receptors proteins have been identified (FZD1, FZD2, FZD3,FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10). In certain embodiments,the Wnt-binding antibody inhibits binding of FZD4, FZD5, and/or FZD8 toone or more Wnts (e.g., Wnt3a). In certain embodiments, the inhibitionof binding of a particular ligand to a Wnt provided by the Wnt-bindingantibody is at least about 10%, at least about 25%, at least about 50%,at least about 75%, at least about 90%, or at least about 95%. Incertain embodiments, an antibody that inhibits binding of a Wnt to aligand such as a FZD, further inhibits Wnt signaling (e g., inhibitscanonical Wnt signaling)

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention inhibits Wnt signaling. It is understood that aWnt-binding antibody that inhibits Wnt signaling can, in certainembodiments, inhibit signaling by one or more Wnts, but not necessarilyby all Wnts. In certain alternative embodiments, signaling by all humanWnts can be inhibited. In certain embodiments, signaling by one or moreWnts selected from the group consisting of Wnt1, Wnt2, Wnt2b/13, Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a(previously Wnt14), Wnt9b (previously Wnt15), Wnt10a, Wnt10b, Wnt11, andWnt16 is inhibited. In certain embodiments, the Wnt signaling that isinhibited is signaling by Wnt1, Wnt2, Wnt3, Wnt3a, Wnt7a, Wnt7b, and/orWnt10b. In certain embodiments, the antibody inhibits signaling by (atleast) Wnt1, Wnt3a, Wnt7b, and Wnt10b. In particular embodiments, theantibody inhibits signaling by (at least) Wnt3a. In certain embodiments,the inhibition of signaling by a Wnt provided by the Wnt-bindingantibody is a reduction in the level of signaling by the Wnt of leastabout 10%, at least about 25%, at least about 50%, at least about 75%,at least about 90%, or at least about 95%. In certain embodiments, theWnt signaling that is inhibited is canonical Wnt signaling.

In vivo and in vitro assays for determining whether a Wnt-bindingantibody inhibits Wnt signaling are known in the art. For example,cell-based, luciferase reporter assays utilizing a TCF/Luc reportervector containing multiple copies of the TCF-binding domain upstream ofa firefly luciferase reporter gene may be used to measure canonical Wntsignaling levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66).The level of Wnt signaling in the presence of one or more Wnts (e.g.,Wnt(s) expressed by transfected cells or provided by Wnt-conditionedmedia) with the Wnt-binding antibody present is compared to the level ofsignaling without the Wnt-binding antibody present. In addition to theTCF/Luc reporter assay, the effect of a Wnt-binding antibody (orcandidate antibody) on canonical Wnt signaling may be measured in vitroor in vivo by measuring the effect of the antibody on the level ofexpression of β-catenin regulated genes, such as c-myc (He et al., 1998,Science, 281:1509-12), cyclin D1 (Tetsu et al., 1999, Nature, 398:422-6)and/or fibronectin (Gradl et al. 1999, Mol. Cell Biol., 19:5576-87). Incertain embodiments, the effect of an antibody on Wnt signaling may alsobe assessed by measuring the effect of the antibody on thephosphorylation state of Dishevelled-1, Dishevelled-2, Dishevelled-3,LRP5, LRP6, and/or β-catenin.

In certain embodiments, the Wnt-binding antibodies useful in the methodsof the invention have one or more of the following effects. Inhibitproliferation of neuroendocrine tumor cells, reduce the tumorigenicityof a neuroendocrine tumor by reducing the frequency of cancer stem cellsin the tumor, inhibit neuroendocrine tumor growth, trigger cell death ofneuroendocrine tumor cells, differentiate neuroendocrine tumorigeniccells to a non-tumorigenic state, prevent metastasis of neuroendocrinetumor cells or decrease survival.

In certain embodiments, the Wnt-binding antibodies useful in the methodsof the invention are capable of inhibiting neuroendocrine tumor growth.In certain embodiments, the Wnt-binding antibodies are capable ofinhibiting neuroendocrine tumor growth in vivo (e.g., in a xenograftmouse model, and/or in a human having cancer).

In certain embodiments, the Wnt-binding antibodies useful in the methodsof the invention are capable of reducing the tumorigenicity of aneuroendocrine tumor. In certain embodiments, the antibody is capable ofreducing the tumorigenicity of a neuroendocrine tumor comprising cancerstem cells in an animal model, such as a mouse xenograft model. Incertain embodiments, the number or frequency of cancer stem cells in aneuroendocrine tumor is reduced by at least about two-fold, aboutthree-fold, about five-fold, about ten-fold, about 50-fold, about100-fold, or about 1000-fold. In certain embodiments, the reduction inthe number or frequency of cancer stem cells is determined by limitingdilution assay using an animal model. Additional examples and guidanceregarding the use of limiting dilution assays to determine a reductionin the number or frequency of cancer stem cells in a tumor can be found,e.g., in International Publication Number WO 2008/042236, U.S. PatentApplication Publication No. 2008/0064049, and U.S. Patent ApplicationPublication No. 2008/0178305, each of which is incorporated by referenceherein in its entirety.

In certain embodiments, the Wnt-binding antibody useful in the methodsof the invention has a circulating half-life in mice, cynomolgousmonkeys, or humans of at least about 5 hours, at least about 10 hours,at least about 24 hours, at least about 3 days, at least about 1 week,or at least about 2 weeks. In certain embodiments, the Wnt-bindingantibody is an IgG (e.g., IgG1 or IgG2) antibody that has a circulatinghalf-life in mice, cynomolgous monkeys, or humans of at least about 5hours, at least about 10 hours, at least about 24 hours, at least about3 days, at least about 1 week, or at least about 2 weeks.

In certain embodiments, an anti-Wnt antibody useful for the methods ofthe invention is a bispecific antibody that specifically recognizes ahuman Wnt. Bispecific antibodies are antibodies that are capable ofspecifically recognizing and binding at least two different epitopes. Inone embodiment, the bispecific anti-Wnt antibody specifically recognizesdifferent epitopes within the same human Wnt. In another embodiment, thebispecific anti-Wnt antibody specifically recognizes different epitopeswithin different human Wnts or on different Wnts.

Alternatively, in certain alternative embodiments, an anti-Wnt antibodyuseful for the methods of the invention is not a bispecific antibody.

In certain embodiments, an anti-Wnt antibody useful for the methods ofthe invention is monospecific. In certain embodiments, each of the oneor more antigen-binding sites that an antibody contains is capable ofbinding (or binds) the same one or more human Wnts. In certainembodiments, an antigen-binding site of the monospecific antibody iscapable of binding (or binds) one, two, three, four, or five (or more)human Wnts.

Anti-Wnt antibodies useful for the methods of the invention aredisclosed in International Publication Number WO 2011/088127, which isincorporated by reference in its entirety.

6. Antibodies and Production Thereof

The antibodies (e.g., anti-FZD and anti-Wnt antibodies) useful in themethods of the invention can be produced by any suitable method known inthe art. Polyclonal antibodies can be prepared by any known method.Polyclonal antibodies are raised by immunizing an animal (e.g. a rabbit,rat, mouse, donkey, etc.) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc.) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g. Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody useful in the methods ofthe invention is a humanized antibody. In certain embodiments, suchantibodies are used therapeutically to reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject. Humanized antibodies can be produced using various techniquesknown in the art. In certain alternative embodiments, the antibodyuseful in the methods of the invention is a human antibody.

Human antibodies can be directly prepared using various techniques knownin the art.

Immortalized human B lymphocytes immunized in vitro or isolated from animmunized individual that produce an antibody directed against a targetantigen can be generated (See, e.g., Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373). Also, the humanantibody can be selected from a phage library, where that phage libraryexpresses human antibodies, as described, for example, in Vaughan etal., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l.Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol.,227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques forthe generation and use of antibody phage libraries are also described inU.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731;6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and7,264,963; and Rothe et al., 2007, J. Mol. Bio.,doi:10.1016/j.jmb.2007.12.018 (each of which is incorporated byreference in its entirety). Affinity maturation strategies and chainshuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783,incorporated by reference in its entirety) are known in the art and maybe employed to generate high affinity human antibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

In certain embodiments, the antibody useful in the methods of theinvention is a bispecific antibody that specifically recognizes a humanfrizzled receptor or a human Wnt polypeptide. Bispecific antibodies areantibodies that are capable of specifically recognizing and binding atleast two different epitopes. The different epitopes can either bewithin the same molecule (e.g. the same human frizzled receptor or samehuman Wnt polypeptide) or on different molecules. Bispecific antibodiescan be intact antibodies or antibody fragments.

Alternatively, in certain alternative embodiments, antibodies useful forthe invention are not bispecific antibodies.

In certain embodiments, the antibodies useful for the invention aremonospecific. For example, in certain embodiments, each of the one ormore antigen-binding sites that an antibody contains is capable ofbinding (or binds) the same human FZD receptor or the same human Wntpolypeptide. In certain embodiments, an antigen-binding site of amonospecific antibody is capable of binding (or binds) one, two, three,four, or five (or more) human frizzled receptors or human Wntpolypeptide.

In certain embodiments, an antibody useful for the methods of theinvention is an antibody fragment. Antibody fragments can displayincreased tumor penetration relative to a full antibody. Varioustechniques are known for the production of antibody fragments.Traditionally, these fragments are derived via proteolytic digestion ofintact antibodies (for example Morimoto et al., 1993, Journal ofBiochemical and Biophysical Methods 24:107-117; Brennan et al., 1985,Science, 229:81). In certain embodiments, antibody fragments areproduced recombinantly. Fab, Fv, and scFv antibody fragments can all beexpressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments. Suchantibody fragments can also be isolated from the antibody phagelibraries discussed above. The antibody fragment can also be linearantibodies as described in U.S. Pat. No. 5,641,870, for example, and canbe monospecific or bispecific. Single-chain antibodies useful in themethods of the invention can be prepared as described, for example, inU.S. Pat. No. 4,946,778. In addition, methods can be adapted for theconstruction of Fab expression libraries (Huse, et al., Science246:1275-1281 (1989)) to allow rapid and effective identification ofmonoclonal Fab fragments with the desired specificity for a FZD receptoror a Wnt polypeptide. Antibody fragments may be produced by techniquesin the art including, but not limited to: (a) a F(ab′)2 fragmentproduced by pepsin digestion of an antibody molecule; (b) a Fab fragmentgenerated by reducing the disulfide bridges of an F(ab′)2 fragment, (c)a Fab fragment generated by the treatment of the antibody molecule withpapain and a reducing agent, and (d) Fv fragments. Other techniques forthe production of antibody fragments will be apparent to the skilledpractitioner.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

In certain embodiments, an antibody useful for the methods of theinvention is a heteroconjugate antibody. Heteroconjugate antibodies arecomposed of two covalently joined antibodies. Such antibodies have, forexample, been proposed to target immune cells to unwanted cells (U.S.Pat. No. 4,676,980). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

It is known in the art that the constant Fc region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to antibodies activates the complement system. Activation ofcomplement is important in the opsonization and lysis of cell pathogens.The activation of complement also stimulates the inflammatory responseand can also be involved in autoimmune hypersensitivity. Further,antibodies or soluble receptors can bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the Wnt antagonist polypeptides (antibodies andFc comprising soluble receptors) useful for the methods of the inventionprovide for altered effector functions that, in turn, affect thebiological profile of the administered polypeptides. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified antibody thereby increasing tumor localization. In other casesit may be that constant region modifications moderate complement bindingand thus reduce the serum half-life and nonspecific association of aconjugated cytotoxin. Yet other modifications of the constant region maybe used to eliminate disulfide linkages or oligosaccharide moieties thatallow for enhanced localization due to increased antigen specificity orantibody flexibility. Similarly, modifications to the constant regionmay easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain embodiments, a Wnt antagonist polypeptide comprising an Fcregion (antibodies and Fc comprising soluble receptors) useful for themethods of the invention does not have one or more effector functions.For instance, in some embodiments, the polypeptide has noantibody-dependent cellular cytotoxicity (ADCC) activity and/or nocomplement-dependent cytotoxicity (CDC) activity. In certainembodiments, the polypeptide does not bind to an Fc receptor and/orcomplement factors. In certain embodiments, the antibody has no effectorfunction.

The invention also pertains to the use of immunoconjugates comprising aWnt antagonist polypeptide (e.g., anti-FZD and anti-Wnt antibody)conjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamycin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies including ²¹²Bi, ¹³¹I, ¹³¹In,⁹⁰Y, and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are madeusing a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Conjugates of an antibody and one ormore small molecule toxins, such as a calicheamicin, maytansinoids, atrichothene, and CC 1065, and the derivatives of these toxins that havetoxin activity, can also be used.

Conjugate antibodies are composed of two covalently joined antibodies.Such antibodies have, for example, been proposed to target immune cellsto unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins can be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Regardless of how useful quantities are obtained, the Wnt antagonistspolypeptides (e.g., antibodies and soluble receptors) useful in themethods of the invention can be used in any one of a number ofconjugated (i.e. an immunoconjugate) or unconjugated forms.Alternatively, the polypeptides can be used in a nonconjugated or“naked” form. In certain embodiments, the polypeptides are used innonconjugated form to harness the subject's natural defense mechanismsincluding complement-dependent cytotoxicity (CDC) and antibody dependentcellular toxicity (ADCC) to eliminate the malignant cells. In someembodiments, the polypeptides can be conjugated to radioisotopes, suchas ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, 111In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu,¹⁸⁶Re and ¹⁸⁸Re using anyone of a number of well-known chelators ordirect labeling. In other embodiments, the compositions can comprise Wntantagonist polypeptides coupled to drugs, prodrugs or biologicalresponse modifiers such as methotrexate, adriamycin, and lymphokinessuch as interferon. Still other embodiments comprise the use of Wntantagonist polypeptides conjugated to specific biotoxins such as ricinor diptheria toxin. In yet other embodiments, the Wnt antagonistpolypeptides can be complexed with other immunologically active ligands(e.g. antibodies or fragments thereof) wherein the resulting moleculebinds to both the neoplastic cell and an effector cell such as a T cell.The selection of which conjugated or unconjugated Wnt antagonistpolypeptides to use will depend of the type and stage of neuroendocrinetumor, use of adjunct treatment (e.g., chemotherapy or externalradiation) and patient condition. It will be appreciated that oneskilled in the art could readily make such a selection in view of theteachings herein.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biologicalhalf-life or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The chemical moieties most suitable for derivatization include watersoluble polymers. A water soluble polymer is desirable because theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. In some embodiments,the polymer will be pharmaceutically acceptable for the preparation of atherapeutic product or composition. One skilled in the art will be ableto select the desired polymer based on such considerations as whetherthe polymer/protein conjugate will be used therapeutically, and if so,the desired dosage, circulation time, resistance to proteolysis, andother considerations. The effectiveness of the derivatization can beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or by injection or infusion, or, further formulated fororal, pulmonary or other delivery routes), and determining itseffectiveness. Suitable water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), dextran, poly(n-vinyl pyrrolidone)-polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde canhave advantages in manufacturing due to its stability in water.

The isolated polypeptides (e.g., antibodies and soluble receptors)useful in the methods of the invention can be produced by any suitablemethod known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express Wnt antagonist polypeptides (e.g., antibodies orsoluble receptors). Recombinant expression vectors are replicable DNAconstructs which have synthetic or cDNA-derived DNA fragments encoding apolypeptide of interest operatively linked to suitable transcriptionalor translational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, transcriptional promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences, as described in detailbelow. Such regulatory elements can include an operator sequence tocontrol transcription. The ability to replicate in a host, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are operatively linked when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovims andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a Wnt antagonist polypeptide(e.g., antibody or soluble receptor) include prokaryotes, yeast, insector higher eukaryotic cells under the control of appropriate promoters.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include establishedcell lines of mammalian origin as described below. Cell-free translationsystems could also be employed. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts are described by Pouwels et al. (Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., 1985), the relevant disclosure of which ishereby incorporated by reference. Additional information regardingmethods of protein production, including antibody production, can befound, e.g., in U.S. Patent Publication No. 2008/0187954, U.S. Pat. Nos.6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is hereby incorporated by reference herein inits entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a Wnt antagonist polypeptide (e.g., antibodyor soluble receptor). Some or all of the foregoing purification steps,in various combinations, can also be employed to provide a homogeneousrecombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying a Wnt antagonist polypeptide(e.g., antibody or soluble receptor) also include, for example, thosedescribed in U.S. Patent Publication No. 2008/0312425, 2008/0177048, and2009/0187005, each of which is hereby incorporated by reference hereinin its entirety.

7. Pharmaceutical Compositions

The Wnt antagonist polypeptides (e.g., antibodies and soluble receptors)can be formulated into a pharmaceutical composition by any suitablemethod known in the art. In certain embodiments, the pharmaceuticalcompositions comprise a pharmaceutically acceptable vehicle. Thepharmaceutical compositions find use in inhibiting neuroendocrine tumorgrowth and treating neuroendocrine tumor in human patients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified Wnt antagonist (e.g., an anti-FZD antibody orsoluble FZD receptor) with a pharmaceutically acceptable vehicle (e.g.carrier, excipient) (Remington, The Science and Practice of Pharmacy20th Edition Mack Publishing, 2000). Suitable pharmaceuticallyacceptable vehicles include, but are not limited to, nontoxic bufferssuch as phosphate, citrate, and other organic acids; salts such assodium chloride; antioxidants including ascorbic acid and methionine;preservatives (e.g. octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl orpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight polypeptides (e.g. less than about 10amino acid residues); proteins such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; carbohydrates such as monosaccharides, disaccharides,glucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and non-ionic surfactants such as TWEEN or polyethyleneglycol (PEG).

In certain embodiments, the pharmaceutical composition is frozen. Incertain alternative embodiments, the pharmaceutical composition islyophilized.

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc. of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The Wnt antagonists (e.g., anti-FZD antibodies or soluble FZD receptors)can also be entrapped in microcapsules. Such microcapsules are prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In certain embodiments, pharmaceutical formulations include the Wntantagonists (e.g., anti-FZD antibodies or soluble FZD receptors)complexed with liposomes (Epstein, et al., 1985, Proc. Natl. Acad. Sci.USA 82:3688; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030;and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Someliposomes can be generated by the reverse phase evaporation with a lipidcomposition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(v nylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1 Neuroendocrine Tumor Response to OMP-18R5 in a Phase 1aClinical Study

In the context of a Phase I clinical trial for the OMP-18R5 humananti-FZD antibody in patients with advanced solid tumors, three patientswith late stage neuroendocrine tumors that had previously undergonemultiple other therapies were treated with low, periodic doses ofOMP-18R5 as a single agent. The prolonged stable disease of all three ofthese neuroendocrine patients suggests that even as a single agent atlow dosages OMP-18R5 may have a surprising level of efficacy againstneuroendocrine tumors, including both neuroendocrine tumors havingcarcinoid histology and pancreatic neuroendocrine tumors.

At the time of her enrollment in the OMP-18R5 trial, Patient 3 was a 59years old female. She was diagnosed with neuroendocrine tumor(carcinoid) in 2004. She underwent a small bowel resection and wastreated with radiofrequency ablation of liver lesions. Prior toenrollment in the OMP-18R5 trial, she received prior systemic treatmentwith a combination of trametinib (MEK1/2 MAP kinase inhibitor) andGSK2141795 Akt inhibitor but her disease progressed after 1 month oftreatment. In the OMP-18R5 trial Patient 3 received a weekly dose of 0.5mg/kg OMP-18R5 for 112 days. Her disease remained stable during OMP-18R5treatment, but she was removed from the trial after suffering a bonefracture on day 112. Especially in light of her rapid diseaseprogression while on a previous therapy, this patient's extended periodof disease control while being treated with OMP-18R5 suggests that theantibody may have a surprising level of clinical efficacy even as asingle agent at a low dose.

At the time of her enrollment in the OMP-18R5 trial, Patient 10 was a 69year old female with pancreatic neuroendocrine tumor. She was diagnosedin 2001 and treated with surgery comprising 80% distal pancreatectomy,splenectomy, and wedge resection of posterior wall of stomach. Prior toenrollment in the OMP-18R5 trial, she received systemic treatments with(1) regorafenib (partial response: 3 years); (2) anti-LOXL2 antibody(stable disease: 5.5 months); and (3) anti-CSFR1 antibody (progressivedisease after 6 weeks on study). As of Jan. 25, 2013, Patient 10 in theOMP-18R5 trial had received 0.5 mg/kg OMP-18R5 every other week for 279days. After 112 days of OMP-18R5 treatment, a 21% reduction in Patient10's target tumor liver metastasis was determined by the investigator.Tumor reduction was confirmed by an independent radiographic assessment(shown in Table 4). See FIG. 1A. The control non-target disease lesionshowed no change during the same treatment period. Radiographicexamination further revealed signs of calcification in the tumor lesionof Patient 10 following 112 days of OMP-18R5 treatment (FIGS. 1B and C).The observed calcification of the tumor lesion may indicate thatOMP-18R5 induced differentiation of the tumor cells and/or tumornecrosis. Subsequent computed tomography (CT) scans on days 168, 224 and280 indicated that the patient still did not have progressive disease.

TABLE 4 Patient 10: Independent Radiographic Assessment RECIST 1.1 Mar.27, 2012 Jun. 11, Aug. 6, Lesion (mm) (BASELINE) 2012 2012 1. Liver: RtLobe 13.6 × 22.9 17.3 × 23.9 13.3 × 20.5 (Ant-Lat) (TARGET) 2. Liver:IVC 16.2 × 16.2 15.9 × 15.9 10.8 × 13.1 (TARGET) 3. Liver: Rt Dome  7.9× 11.6  7.1 × 11.4 4.9 × 8.5 (TARGET) 4. Porto-caval Node 16.6 × 23.914.1 × 15.5  9.1 × 14.2 (TARGET)* 5. Porto-caval Node 11.1 × 14.5 10.5 ×12.6  9.5 × 14.8 (NON-TARGET) TOTAL: Target (mm, % Δ) 67.3 65.3 (−3%)51.2 (−24%) TOTAL: Non-Target Non-PD Non-PD Normal** *Per RECIST 1.1: LN≧ 15 mm in shortest diameter are measurable **Per RECIST 1.1: LN < 10 mmin shortest diameter considered ‘normal’ Non-PD: non-progressive disease

At the time of her enrollment in the OMP-18R5 trial, Patient 12 was a 77year old female with a neuroendocrine tumor (carcinoid). She wasdiagnosed in 2006. Prior to enrollment in the OMP-18R5 trial, shereceived systemic treatments with (1) sandostatin (stable disease: 20months); (2) inhibitor of heat shock protein 90 (stable disease: 23months); and (3) a combination of sandostatin and anti-angiopoietin-2antibody (stable disease: 4 months). As of Jan. 25, 2013, Patient 12 inthe OMP-18R5 trial had received lmg/kg OMP-18R5 every third week for 210days. This patient was assessed to have stable disease on days 56, 112and 168. The extended period of time during which this patient hasremained on the clinical trial without disease progression furthersupports the clinical efficacy of OMP-18R5 against neuroendocrinetumors.

FIG. 2 shows the number of days that each of the patients (n=18)enrolled in the OMP-18R5 Phase 1a study as of Jan. 25, 2013, stayed onthe OMP-18R5 Phase 1a study. The patients with neuroendocrine tumorsthat had been treated with OMP-18R5 remained on study for surprisinglylong periods of times relative to the other Phase 1a patients havingother tumor types (including colorectal cancer, breast cancer, melanomaand pancreatic cancer).

Also, as of Jan. 25, 2013, the three patients with neuroendocrine tumorshad had stable disease ˜2- to 7-fold longer on OMP-18R5 treatment thanwhen they were on the prior therapies on which they previouslyprogressed. Using Growth Modulation Index as a tool to gauge theobserved activity (time on current therapy divided by time on priortherapy before progressive disease; GMI≧1.33 considered excellent; VonHoff; Clinical Cancer Research 4:1079-1086, 1998), all threeneuroendocrine (NET) patients significantly surpassed this mark (Patient12: 1.8; Patient 10: 6.3; Patient 3, off study: 3.8). A comparison ofthe time each of the three neuroendocrine tumor patients remained on theOMP-18R5 study (as of Jan. 25, 2013) versus her time on prior therapiesis shown in FIG. 3.

Example 2 In vivo Prevention of Neuroendocrine Tumor Growth Using a WntAntagonist

This example describes a use of a Wnt antagonist (e.g., OMP-18R5 orOMP-54F28) to prevent neuroendocrine tumor growth in a xenograft model.In certain embodiments, neuroendocrine tumor cells from a patient sample(solid tumor biopsy or pleural effusion) that have been passaged as axenograft in mice are prepared for repassaging into experimentalanimals. Neuroendocrine tumor tissue is removed under sterileconditions, cut up into small pieces, minced completely using sterileblades, and single cell suspensions obtained by enzymatic digestion andmechanical disruption. Specifically, pleural effusion cells or theresulting tumor pieces are mixed with ultra-pure collagenase III inculture medium (200-250 units of collagenase per mL) and incubated at37° C. for 3-4 hours with pipetting up and down through a 10 mL pipetteevery 15-20 minutes. Digested cells are filtered through a 45 μM nylonmesh, washed with RPMI/20% FBS, and washed twice with HBSS. Dissociatedneuroendocrine tumor cells are then injected subcutaneously into themammary fat pads of NOD/SCID mice to elicit tumor growth.

In certain embodiments, dissociated neuroendocrine tumor cells are firstsorted into tumorigenic and non-tumorigenic cells based on cell surfacemarkers before injection into experimental animals. Specifically,neuroendocrine tumor cells dissociated as described above are washedtwice with Hepes buffered saline solution (HBSS) containing 2%heat-inactivated calf serum (HICS) and resuspended at 10⁶ cells per 100μl. Antibodies are added and the cells incubated for 20 minutes on icefollowed by two washes with HBSS/2% HICS. Antibodies include anti-ESA(Biomeda, Foster City, Calif.), anti-CD44, anti-CD24, and Lineagemarkers anti-CD2, -CD3, -CD10, -CD16, -CD18, -CD31, -CD64, and -CD140b(collectively referred to as Lin; PharMingen, San Jose, Calif.).Antibodies are directly conjugated to fluorochromes to positively ornegatively select cells expressing these markers. Mouse cells areeliminated by selecting against H2Kd+ cells, and dead cells areeliminated by using the viability dye 7AAD. Flow cytometry is performedon a FACSVantage (Becton Dickinson, Franklin Lakes, N.J.). Side scatterand forward scatter profiles are used to eliminate cell clumps. IsolatedESA+, CD44+, CD24-/low, Lin-tumorigenic cells are then injectedsubcutaneously into NOD/SCID mice to elicit tumor growth.

By way of example, Wnt antagonists (e.g., OMP-18R5 or OMP-54F28) areanalyzed for their ability to reduce the growth of neuroendocrine tumorcells. Dissociated neuroendocrine tumor cells (10,000 per animal) areinjected subcutaneously into the flank region of 6-8 week old NOD/SCIDmice. Two days after tumor cell injection, animals are injectedintraperitoneal (i.p.) with 10 mg/kg anti-FZD antibody or soluble FZDreceptor two times per week. Tumor growth is monitored weekly untilgrowth is detected, after which point tumor growth is measured twiceweekly for a total of 8 weeks. FZD-binding antibodies whichsignificantly reduce tumor growth as compared to PBS injected controlsare thus identified.

Example 3 In vivo Treatment of Neuroendocrine Tumors Using a WntAntagonist

This example describes the use of a Wnt antagonists (e.g., OMP-18R5 orOMP-54F28) to treat neuroendocrine cancer in a xenograft model. Incertain embodiments, neuroendocrine tumor cells from a patient sample(solid tumor biopsy or pleural effusion) that have been passaged as axenograft in mice are prepared for repassaging into experimentalanimals. Neuroendocrine tumor tissue is removed, cut up into smallpieces, minced completely using sterile blades, and single cellsuspensions obtained by enzymatic digestion and mechanical disruption.Dissociated neuroendocrine tumor cells are then injected subcutaneouslyeither into the mammary fat pads, for breast tumors, or into the flank,for non-breast tumors, of NOD/SCID mice to elicit tumor growth.Alternatively, ESA+, CD44+, CD24-/low, Lin-tumorigenic tumor cells areisolated as described above and injected.

Following tumor cell injection, animals are monitored for tumor growth.Once neuroendocrine tumors reach an average size of approximately 150 to200 mm, Wnt antagonist (e.g., OMP-18R5 or OMP-54F28) treatment begins.Each animal receives 100 μg Wnt antagonist (e.g., OMP-18R5 or OMP-54F28)or control agents i.p. two to five times per week for a total of 6weeks. Tumor size is assessed twice a week during these 6 weeks. Theability of Wnt antagonists (e.g., OMP-18R5 or OMP-54F28) to preventfurther neuroendocrine tumor growth or to reduce neuroendocrine tumorsize compared to control agents is thus determined.

At the end point of antibody treatment, tumors are harvested for furtheranalysis. In some embodiments a portion of the neuroendocrine tumor isanalyzed by immunofluorescence to assess Wnt antagonist (e.g., OMP-18R5or OMP-54F28) penetration into the tumor and tumor response. A portionof each harvested neuroendocrine tumor from Wnt antagonist (e.g.,OMP-18R5 or OMP-54F28) treated and control mice is fresh-frozen inliquid nitrogen, embedded in OCT., and cut on a cryostat as 10 μmsections onto glass slides. In some embodiments, a portion of eachneuroendocrine tumor is formalin-fixed, paraffin-embedded, and cut on amicrotome as 10 μm section onto glass slides. Sections are post-fixedand incubated with chromophore labeled antibodies that specificallyrecognize the injected Wnt antagonist (e.g., OMP-18R5 or OMP-54F28) todetect Wnt antagonists (e.g., OMP-18R5 or OMP-54F28) or control agentspresent in the tumor biopsy. Furthermore antibodies that detectdifferent tumor and tumor-recruited cell types such as, for example,anti-VE cadherin (CD144) or anti-PECAM-1 (CD31) antibodies to detectvascular endothelial cells, anti-smooth muscle alpha-actin antibodies todetect vascular smooth muscle cells, anti-Ki67 antibodies to detectproliferating cells, TUNEL assays to detect dying cells, anti-β-cateninantibodies to detect Wnt signaling, and anti-intracellular domain (ICD)Notch fragment antibodies to detect Notch signaling can be used toassess the effects of Wnt antagonist (e.g., OMP-18R5 or OMP-54F28)treatment on, for example, angiogenesis, tumor growth and tumormorphology.

In certain embodiments, the effect of Wnt antagonist (e.g., OMP-18R5 orOMP-54F28) treatment on neuroendocrine tumor cell gene expression isalso assessed. Total RNA is extracted from a portion of each harvestedneuroendocrine tumor from Wnt antagonist (e.g., OMP-18R5 or OMP-54F28)treated and control antibody treated mice and used for quantitativeRT-PCR. Expression levels of FZD receptors, components of Wnt signalingpathway including, for example, Wnt1 and β-catenin, as well asadditional cancer stem cell markers previously identified (e.g. CD44)are analyzed relative to the housekeeping gene GAPDH as an internalcontrol. Changes in neuroendocrine tumor cell gene expression upontreatment with Wnt antagonists (e.g., OMP-18R5 or OMP-54F28) are thusdetermined.

In addition, the effect of Wnt antagonist (e.g., OMP-18R5 or OMP-54F28)treatment on the frequency of cancer stem cells in a neuroendocrinetumor is assessed. Neuroendocrine tumor samples from Wnt antagonist(e.g., OMP-18R5 or OMP-54F28) versus control agent treated mice are cutup into small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. Dissociated neuroendocrine tumor cells are then analyzed byFACS analysis for the presence of tumorigenic cancer stem cells based onESA+, CD44+, CD24-/low, Lin-surface cell marker expression as describedin detail above.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24-/low,Lin-expression following Wnt antagonist (e.g., OMP-18R5 or OMP-54F28)treatment can then assessed. ESA+, CD44+, CD24-/low, Lin-cancer stemcells isolated from Wnt antagonist (e.g., OMP-18R5 or OMP-54F28) treatedversus control agent treated mice are re-injected subcutaneously intothe mammary fat pads of NOD/SCID mice. The tumorigenicity of cancer stemcells based on the number of injected cells required for consistentneuroendocrine tumor formation is then determined.

Example 4 Treatment of Human Neuroendocrine Tumor Using Anti-FZDReceptor Antibodies or Soluble FZD Receptors

This example describes certain methods for treating neuroendocrine tumorusing antibodies against a FZD receptor to target neuroendocrine tumorscomprising cancer stem cells and/or tumor cells in which FZD receptorexpression has been detected and/or tumor cells having a Wnt genesignature indicating that they are responsive to inhibition of Wntsignaling.

In some embodiments, the presence of cancer stem cell marker or FZDreceptor or the expression of one or more genes in a Wnt gene signaturecan first be determined from a tumor biopsy. Tumor cells from a biopsyfrom a patient diagnosed with neuroendocrine tumor are removed understerile conditions. In some embodiments the tissue biopsy isfresh-frozen in liquid nitrogen, embedded in OCT., and cut on a cryostatas 10 μm sections onto glass slides. In some embodiments, the tissuebiopsy is formalin-fixed, paraffin-embedded, and cut on a microtome as10um section onto glass slides.

Sections are incubated with antibodies against a FZD receptor to detectFZD protein expression. Alternatively, sections can be analyzed for thepresence of one or more genes in the Wnt gene signature.

The presence of cancer stem cells also may be determined. Tissue biopsysamples are cut up into small pieces, minced completely using sterileblades, and cells subject to enzymatic digestion and mechanicaldisruption to obtain a single cell suspension. Dissociatedneuroendocrine tumor cells are then incubated with anti-ESA, -CD44,-CD24, -Lin, and -FZD antibodies to detect cancer stem cells, and thepresence of ESA+, CD44+, CD24-/low, Lin-, FZD+tumor stem cells isdetermined by flow cytometry as described in detail above.

Cancer patients whose neuroendocrine tumors are diagnosed as expressinga FZD receptor and/or one or more genes in the Wnt gene signature aretreated with anti-FZD receptor antibodies or soluble FZD receptors. Incertain embodiments, humanized or human monoclonal anti-FZD receptorantibodies or soluble FZD receptors are purified and formulated with asuitable pharmaceutical vehicle for injection. In some embodiments,patients are treated with the FZD antibodies or soluble FZD receptors atleast once a month for at least 10 weeks. In some embodiments, patientsare treated with the FZD antibodies or soluble FZD receptors at leastonce a week for at least about 14 weeks. Each administration of theantibody or soluble FZD receptors should be a pharmaceutically effectivedose. In some embodiments, between about 2 to about 100 mg/ml of ananti-FZD antibody or soluble FZD receptors is administered. In someembodiments, between about 5 to about 40 mg/ml of an anti-FZD antibodyor soluble FZD receptors is administered. The antibody or soluble FZDreceptors can be administered prior to, concurrently with, or afterstandard radiotherapy regimens or chemotherapy regimens using one ormore chemotherapeutic agent. Patients are monitored to determine whethersuch treatment has resulted in an anti-tumor response, for example,based on tumor regression, reduction in the incidences of new tumors,lower tumor antigen expression, decreased numbers of cancer stem cells,or other means of evaluating disease prognosis.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein, as well as U.S. Ser. No.61/717,294, filed Oct. 23, 2012, are hereby incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, internet site, or accessionnumber/database sequence were specifically and individually indicated tobe so incorporated by reference.

SEQUENCES Human FZD1 full length amino acid sequence (SEQ ID NO: 1; underlining indicates ECD):MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAGFLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEATEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVIACYFYEQAFRDQWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSGKTLNSWRKFYTRLTNSKQGETTVHuman FZD2 full length amino acid sequence (SEQ ID NO: 2; underlining indicates ECD):MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEETRFARLWILTWSVLCCASTFFTVTTYLVDMQRFRYPERPIIFLSGCYTMVSVAYIAGFVLQERVVCNERFSEDGYRTVVQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEATEANSQYFHLAAWAVPAVKTITILAMGQIDGDLLSGVCFVGLNSLDPLRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLERLMVRIGVFSVLYTVPATIVIACYFYEQAFREHWERSWVSQHCKSLAIPCPAHYTPRMSPDFTVYMIKYLMTLIVGITSGFWIWSGKTLHSWRK FYTRLTNSRHGETTVHuman FZD3 full length amino acid sequence  (SEQ ID NO: 3):MAMTWIVFSLWPLTVFMGHIGGHSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLNLAGEPTEGAPVAVQRDYGFWCPRELKIDPDLGYSFLHVRDCSPPCPNMYFRREELSFARYFIGLISIICLSATLFTFLTFLIDVTRFRYPERPIIFYAVCYMMVSLIFFIGFLLEDRVACNASIPAQYKASTVTQGSHNKACTMLFMILYFFTMAGSVWWVILTITWFLAAVPKWGSEATEKKALLFHASAWGIPGILTIILLAMNKIEGDNISGVCFVGLYDVDALRYFVLAPLCLYVVVGVSLLLAGIISLNRVRIEIPLEKENQDKLVKFMIRIGVFSILYLVPLLVVIGCYFYEQAYRGIWETTWIQERCREYHIPCPYQVTQMSRPDLILFLMKYLMALIVGIPSVFWVGSKKTCFEWASFFHGRRKKEIVNESRQVLQEPDFAQSLLRDPNTPIIRKSRGTSTQGTSTHASSTQLAMVDDQRSKAGSIHSKVSSYHGSLHRSRDGRYTPCSYRGMEERLPHGSMSRLTDHSRHSSSHRLNEQSRHSSIRDLSNNPMTHIT HGTSMNRVIEEDGTSAHuman FZD4 full length amino acid sequence  (SEQ ID NO: 4):MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEFTDIWMAVWASLCFISTAFTVLTFLIDSSRFSYPERPIIFLSMCYNIYSIAYIVRLTVGRERISCDFEEAAEPVLIQEGLKNIGCATIFLLMYFFGMASSIWWVILTLTWFLAAGLKWGHEATEMHSSYFHIAAWAIPAVKTIVILIMRLVDADELTGLCYVGNQNLDALTGFVVAPLFTYLVIGTLFIAAGLVALFKIRSNLQKDGTKTDKLERLMVKIGVFSVLYTVPATCVIACYFYEISNWALFRYSADDSNMAVEMLKIFMSLLVGITSGMWIWSAKTLHTWQKCSNRLVNSGKVKREKRGNGWVKPGKGSETVHuman FZD5 full length amino acid sequence (SEQ ID NO: 5; underlining indicates ECD):MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERTFATFWIGLWSVLCFISTSTTVATFLIDMERFRYPERPIIFLSACYLCVSLGFLVRLVVGHASVACSREHNHIHYETTGPALCTIVFLLVYFFGMASSIWWVILSLTWFLAAGMKWGNEATAGYAQYFHLAAWLIPSVKSITALALSSVDGDPVAGICYVGNQNLNSLRGFVLGPLVLYLLVGTLFLLAGFVSLFRIRSVIKQGGTKTDKLEKLMIRIGIFTLLYTVPASIVVACYLYEQHYRESWEAALICACPGHDTGQPRAKPEYWVLMLKYFMCLVVGITSGVWIWSGKIVESWRRFTSRCCCRPRRGHKSGGAMAAGDYPEASAALTGRTGPPGPAATYHKQVSLSHVHuman FZD6 full length amino acid sequence (SEQ ID NO: 6; underlining indicates ECD):MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELEFAKSFIGTVSIFCLCATLFTFLTFLIDVRRFRYPERPITYYSVCYSIVSLMYFIGFLLGDSTACNKADEKLELGDTVVLGSQNKACTVLFMLLYFFTMAGTVWWVILTITWFLAAGRKWSCEATEQKAVWFHAVAWGTPGFLTVMLLAMNKVEGDNISGVCFVGLYDLDASRYFVLLPLCLCVFVGLSLLLAGIISLNHVRQVIQHDGRNQEKLKKFMIRIGVFSGLYLVPLVILLGCYVYEQVNRITWEITWVSDHCRQYHIPCPYQAKAKARPELALFMIKYLMTLIVGISAVFWVGSKKTCTEWAGFFKRNRKRDPISESRRVLQESCEFFLKHNSKVKHKKKHYKPSSHKLKVISKSMGTSTGATANHGTSAVAITSHDYLGQETLTEIQTSPETSMREVKADGASTPRLREQDCGEPASPAASISRLSGEQVDGKGQAGSVSESARSEGRISPKSDITDTGLAQSNNLQVPSSSEPSSLKGSTSLLVHPVSGVRKEQGG GCHSDTHuman FZD7 full length amino acid sequence (SEQ ID NO: 7; ECD is underlined):MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARLWVGVWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYFMVAVAHVAGFLLEDRAVCVERFSDDGYRTVAQGTKKEGCTILFMVLYFFGMASSIWWVILSLTWFLAAGMKWGHEATEANSQYFHLAAWAVPAVKTITILAMGQVDGDLLSGVCYVGLSSVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVLACYFYEQAFREHWERTWLLQTCKSYAVPCPPGHFPPMSPDFTVFMIKYLMTMIVGITTGFWIWSGKTLQSWRRFYHRLSHSSKGETAV Human FZD8 full length amino acid sequence (SEQ ID NO: 8; ECD is underlined):MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPRRLPPPPPGEQPPSGSGHGRPPGARPPHRGGGRGGGGGDAAAPPARGGGGGGKARPPGGGAAPCEPGCQCRAPMVSVSSERHPLYNRVKIGQIANCALPCHNPFFSQDERAFTVFWIGLWSVLCFVSTFATVSTFLIDMERFKYPERPIIFLSACYLFVSVGYLVRLVAGHEKVACSGGAPGAGGAGGAGGAAAGAGAAGAGAGGPGGRGEYEELGAVEQHVRYETTGPALCTVVFLLVYFFGMASSIWWVILSLTWFLAAGMKWGNEATAGYSQYFHLAAWLVPSVKSIAVLALSSVDGDPVAGICYVGNQSLDNLRGFVLAPLVIYLFIGTMFLLAGFVSLFRIRSVIKQQDGPTKTHKLEKLMIRLGLFTVLYTVPAAVVVACLFYEQHNRPRWEATHNCPCLRDLQPDQARRPDYAVFMLKYFMCLVVGITSGVWVWSGKTLESWRSLCTRCCWASKGAAVGGGAGATAAGGGGGPGGGGGGGPGGGGGPGGGGGSLYSDVSTGLTWRSGTASSVSYPKQMPLSQVHuman FZD9 full length amino acid sequence  (SEQ ID NO: 9):MAVAPLRGALLLWQLLAAGGAALEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATAGPAEPHKGLGMLPVAPRPARPPGDLGPGAGGSGTCENPEKFQYVEKSRSCAPRCGPGVEVFWSRRDKDFALVWMAVWSALCFFSTAFTVLTFLLEPHRFQYPERPIIFLSMCYNVYSLAFLIRAVAGAQSVACDQEAGALYVIQEGLENTGCTLVFLLLYYFGMASSLWWVVLTLTWFLAAGKKWGHEATEAHGSYFHMAAWGLPALKTIVILTLRKVAGDELTGLCYVASTDAAALTGFVLVPLSGYLVLGSSFLLTGFVALFHIRKIMKTGGTNTEKLEKLMVKIGVFSILYTVPATCVIVCYVYERLNMDFWRLRATEQPCAAAAGPGGRRDCSLPGGSVPTVAVFMLKIFMSLVVGITSGVWVWSSKTFQTWQSLCYRKIAAGRARAKACRAPGSYGRGTHCHYKAPTVVLHMTKTDPSLENPTHLHuman FZD10 full length amino acid sequence (SEQ ID NO: 10; ECD is underlined):MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAATQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKRFAVVWLAIWAVLCFFSSAFTVLTFLIDPARFRYPERPIIFLSMCYCVYSVGYLIRLFAGAESIACDRDSGQLYVIQEGLESTGCTLVFLVLYYFGMASSLWWVVLTLTWFLAAGKKWGHEATEANSSYFHLAAWAIPAVKTILILVMRRVAGDELTGVCYVGSMDVNALTGFVLIPLACYLVIGTSFILSGFVALFHIRRVMKTGGENTDKLEKLMVRIGLFSVLYTVPATCVIACYFYERLNMDYWKILAAQHKCKMNNQTKTLDCLMAASIPAVEIFMVKIFMLLVVGITSGMWIWTSKTLQSWQQVCSRRLKKKSRRKPASVITSGGIYKKAQHPQKTHHGKYEIPAQSPTCV Human FZD1 ECD with signal sequence (SEQ ID NO: 11): MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPC EPTKVYGLMYFGPEELRFSRTHuman FZD2 ECD with signal sequence  (SEQ ID NO: 12):MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEETRFARLWILTHuman FZD3 ECD with signal sequence  (SEQ ID NO: 13):MAMTWIVFSLWPLTVFMGHIGGHSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLNLAGEPTEGAPVAVQRDYGFWCPRELKIDPDLGYSFLHVRDCSPPCPNMYFRREELS FARYHuman FZD4 ECD with signal sequence  (SEQ ID NO: 14):MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEFTDI Human FZD5 ECD with signal sequence (SEQ ID NO: 15): MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT Human FZD6 ECD with signal sequence (SEQ ID NO: 16): MEMFTFLLICIFLPLLRGHSLFICEPITVPRCMKMAYNMIFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHIEFLGPQKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELEFAKS FIGTVSIHuman FZD7 ECD with signal sequence  (SEQ ID NO: 17):MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCIDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCIVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEER RFARLHuman FZD8 ECD with signal sequence  (SEQ ID NO: 18):MEWGYLLEVISLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITAAPSPPRRLPPPPPGEQPPSGSGHGRPPGARPPHRGGGRGGGGGDAAAPPARGGGGGGKARPPGGGAAPCEPGCQCRAPMVSVSSERHPLYNRVKTGQIANCALPCHNPFFSQDERAFT Human FZD9 ECD with signal sequence (SEQ ID NO: 19): MAVAPLRGALLLWQLLAAGGAALEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLIRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCIDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATAGPAEPHKGLGMLPVAPRPARPPGDLGPGAGGSGICENPEKFQYVEKSRSCAPRCGPGVEVFWSRRDKDF Human FZD10 ECD with signal sequence (SEQ ID NO: 20): MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMIRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCIPGVDVYWSREDKRFAHuman FZD1 Fri domain amino acid sequence (SEQID NO: 21; amino acids 87-237 of SEQ ID NO: 1):QQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCIVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKG THuman FZD2 Fri domain amino acid sequence (SEQ ID NO: 22; amino acids 24-159 of SEQ ID NO: 2):QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCIVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGHuman FZD3 Fri domain amino acid sequence (SEQ ID NO: 23; amino acids 23-143 of SEQ ID NO: 3):HSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVILPCRRLCQRAYSECSKLMEMFGVPMP EDMECSRFPDCDEPYPRLVDLHuman FZD4 Fri domain amino acid sequence (SEQ ID NO: 24; amino acids 40-170 of SEQ ID NO: 4):FGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVHuman FZD5 Fri domain amino acid sequence (SEQ ID NO: 25; amino acids 27-157 of SEQ ID NO: 5):ASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAMPERMSCDRLPVLGRDAEVLCMDYNRSEATTHuman FZD6 Fri domain amino acid sequence (SEQ ID NO: 26; amino acids 19-146 of SEQ ID NO: 6):HSLFICEPITVPRCMKMAYNMIFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGHuman FZD7 Fri domain amino acid sequence (SEQ ID NO: 27; amino acids 33-170 of SEQ ID NO: 7):QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCIVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGHuman FZD8 Fri domain amino acid sequence (SEQ ID NO: 28; amino acids 28-158 of SEQ ID NO: 8):ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITHuman FZD9 Fri domain amino acid sequence (SEQ ID NO: 29; amino acids 23-159 of SEQ ID NO: 9):LEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLIRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCIDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENAHuman FZD10 Fri domain amino acid sequence (SEQ ID NO: 30; amino acids 21-154 of SEQ ID NO: 10):ISSMDMERPGDGKCQPIEIPMCKDIGYNMIRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNG 18R5 VH CDR1  (SEQ ID NO: 31)GFTFSHYTLS 18R5 VH CDR2  (SEQ ID NO: 32) VISGDGSYTYYADSVKG 18R5 VH CDR3 (SEQ ID NO: 33) NFIKYVFAN 18R5 VL CDR1  (SEQ ID NO: 34) SGDNIGSFYVH18R5 VL CDR2  (SEQ ID NO: 35) DKSNRPSG 18R5 VL CDR3  (SEQ ID NO: 36)QSYANTLSL 18R5 VH  (SEQ ID NO: 37)EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYTLSWVRQAPGKGLEWVSVISGDGSYTYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCARNF IKYVFANWGQGTLVIVSS18R5 VL  (SEQ ID NO: 38)DIELTQPPSVSVAPGQTARISCSGDNIGSFYVHWYQQKPGQAPVLVIYDKSNRPSGIPERFSGSNSGNTAILTISGTQAEDEADYYCQSYANTLSLVEGG GIKLIVLG18R5 heavy chain (IgG2) amino acid sequence,  underlining indicates VH (SEQ ID NO: 39) MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFSHYTLSWVRQAPGKGLEWVSVISGDGSYTYYADSVKGRFTISSDNSKNTLYLQMNSLRAEDTAVYYCARNFIKYVFANWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK18R5 LIGHT CHAIN light chain (lambda) amino acid sequence, underlining indicates VL  (SEQ ID NO: 40)MAWALLLLTLLTQGTGSWADIELTQPPSVSVAPGQTARISCSGDNIGSFYVHWYQQKPGQAPVLVIYDKSNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQSYANTLSLVEGGGIKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 18R8 VL CDR1  (SEQ ID NO: 41)SGDKLGKKYAS 18R8 VL CDR2  (SEQ ID NO: 42) EKDNRPSG 18R8 VL CDR3 (SEQ ID NO: 43) SSFAGNSLE 18R8 VL  (SEQ ID NO: 44)DIELTQPPSVSVAPGQTARISCSGDKLGKKYASWYQQKPGQAPVLVIYEKDNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSFAGNSLEVFGG GTKLTVLG18R8 18R8 light chain (lambda) amino acidsequence, underlining indicates VL  (SEQ ID NO: 45)MAWALLLLTLLTQGTGSWADIELTQPPSVSVAPGQTARISCSGDKLGKKYASWYQQKPGQAPVLVIYEKDNRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSSFAGNSLEVEGGGIKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 44R24 VH CDR1  (SEQ ID NO: 46)GFTFSSYYIT 44R24 VH CDR2  (SEQ ID NO: 47) TISYSSSNTYYADSVKG44R24 VH CDR3  (SEQ ID NO: 48) SIVFDY 44R24 VL CDR1  (SEQ ID NO: 49)SGDALGNRYVY 44R24 VL CDR2  (SEQ ID NO: 50) SG 44R24 VL CDR3 (SEQ ID NO: 51) GSWDTRPYPKY 44R24 VH  (SEQ ID NO: 52)EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYYITWVRQAPGKGLEWVSTISYSSSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSI VEDYWGQGTLVIVSS44R24 VL  (SEQ ID NO: 53)DIELTQPPSVSVAPGQTARISCSGDALGNRYVYWYQQKPGQAPVLVIPSGIPERFSGSNSGNTAILTISGTQAEDEADYYCGSWDIRPYPKYVFGGGIKL IVLG SEQ ID NO: 54CPLYFPLYC SEQ ID NO: 55 CPLVWPLIC SEQ ID NO: 56 CPLAWPLIC SEQ ID NO: 57CPVKYPLVC SEQ ID NO: 58 CPLRFPLFC SEQ ID NO: 59 CPLAWPLIC SEQ ID NO: 60CPVKFPLYC SEQ ID NO: 61 CPVNYPLYC SEQ ID NO: 62 CPVKFPLYC SEQ ID NO: 63CPLTYPLYC SEQ ID NO: 64 CPLRWPLMC SEQ ID NO: 65 CPLQYPLMC SEQ ID NO: 66CPLSFPLYC SEQ ID NO: 67 CPLNWPLMC SEQ ID NO: 68CP(L/V)X(Y/F/W)PL(Y/F/I/V/M)C SEQ ID NO: 69 DTLSALIERGLM SEQ ID NO: 70DVWWLGSTWLKR SEQ ID NO: 71 FGNYLNDVRFLI SEQ ID NO: 72 TNLADIAHWISGMinimum FZD and SFRP Fri domain sequences h-FZD1 amino acids 116-227 (SEQ ID NO: 73) CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCIVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKC EKFPVHGAGELCh-FZD2 amino acids 39-150  (SEQ ID NO: 74)CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCIVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRC EHFPRHGAEQICh-FZD3 amino acids 28-133  (SEQ ID NO: 75)CEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMEC SRFPDCh-FZD4 amino acids 48-161  (SEQ ID NO: 76)CDPIRISMCQNLGYNVIKMPNLVGHELQTDAELQLTIFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLN CSKFPPQNDHNHMCh-FZD5 amino acids 33-147  (SEQ ID NO: 77)CQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAMPERMS CDRLPVLGRDAEVLCh-FZD6 amino acids 24-129  (SEQ ID NO: 78)CEPITVPRCMKMAYNMIFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELEC DRLQYCh-FZD7 amino acids 49-160  (SEQ ID NO: 79)CQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCIVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRC ENFPVHGAGEICh-FZD8 amino acids 35-148  (SEQ ID NO: 80)CQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMR CDRLPEQGNPDTLCh-FZD9 amino acids 39-152  (SEQ ID NO: 81)CQAVEIPMCRGIGYNLIRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCIDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLD CARLPTRNDPHALCh-FZD10 amino acids 34-147  (SEQ ID NO: 82)CQPIEIPMCKDIGYNMIRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLD CRKLPNKNDPNYLCh-SFRP1 amino acids 57-165  (SEQ ID NO: 83)CVDIPADLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPLLNKNCHAGTQVFLCSLFAPVCLDRPIYPCRWLCEAVRDSCEPVMQFFGFYWPEMLKC DKFPEGDVCh-SFRP2 amino acids 40-152  (SEQ ID NO: 84)CKPIPANLQLCHGIEYQNMRLPNLLGHETMKEVLEQAGAWIPLVMKQCHPDTKKFLCSLFAPVCLDDLDETIQPCHSLCVQVKDRCAPVMSAFGFPWPDM LECDRFPQDNDLCh-SFRP3 amino acids 35-147  (SEQ ID NO: 85)CEPVRIPLCKSLPWNMTKMPNHLHHSTQANAILAIEQFEGLLGTHCSPDLLFFLCAMYAPICTIDFQHEPIKPCKSVCERARQGCEPILIKYRHSWPENL ACEELPVYDRGVCh-SFRP4 amino acids 24-136  (SEQ ID NO: 86)CEAVRIPMCRHMPWNITRMPNHLHHSTQENAILAIEQYEELVDVNCSAVLRFFFCAMYAPICTLEFLHDPIKPCKSVCQRARDDCEPLMKMYNHSWPESL ACDELPVYDRGVCh-SFRP5 amino acids 53-162  (SEQ ID NO: 87)CLDIPADLPLCHTVGYKRMRLPNLLEHESLAEVKQQASSWLPLLAKRCHSDIQVFLCSLFAPVCLDRPIYPCRSLCEAVRAGCAPLMEAYGFPWPEMLHC HKFPLDNDLCh-ROR1 minimal Fri domain  (SEQ ID NO: 88)CQPYRGIACARFIGNRIVYMESLHMQGEIENQITAAFTMIGISSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARS NPMILMRLKLPNCEDLPQPESPEAANC h-ROR2 minimal Fri domain  (SEQ ID NO: 89)CQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGISTHLSDQCSQFAIPSFCHFVFPLCDARSRIPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANCHuman FZD4 Fri domain (predicted signal sequence  underlined)(SEQ ID NO: 90) MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSK FPPQNDHNHMCMEGPGDEEVHuman FZD5 Fri domain (predicted signal sequence  underlined)(SEQ ID NO: 91) MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDY NRSEATTHuman FZD8 Fri domain (predicted signal sequence  underlined)(SEQ ID NO: 92) MEWGYLLEVISLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMD YNRTDLTTHuman IgG1 Fc region  (SEQ ID NO: 93)DKIHTCPPCPAPELLGGPSVFLFPPKPKDILMISRIPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region  (SEQ ID NO: 94)KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region  (SEQ ID NO: 95)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Linker  (SEQ ID NO: 96) ESGGGGVTLinker  (SEQ ID NO: 97) LESGGGGVT Linker  (SEQ ID NO: 98) GRAQVT Linker (SEQ ID NO: 99) WRAQVT Linker  (SEQ ID NO: 100) ARGRAQVTSignal Sequence  (SEQ ID NO: 101) MEWGYLLEVTSLLAALALLQRSSGAAASignal Sequence  (SEQ ID NO: 102) MEWGYLLEVTSLLAALALLQRSSGALASignal Sequence  (SEQ ID NO: 103) MEWGYLLEVTSLLAALALLQRSSGVLASignal Sequence  (SEQ ID NO: 104) MEWGYLLEVTSLLAALLLLQRSPIVHASignal Sequence  (SEQ ID NO: 105) MEWGYLLEVTSLLAALFLLQRSPIVHASignal Sequence  (SEQ ID NO: 106) MEWGYLLEVTSLLAALLLLQRSPFVHASignal Sequence  (SEQ ID NO: 107) MEWGYLLEVTSLLAALLLLQRSPIIYASignal Sequence  (SEQ ID NO: 108) MEWGYLLEVTSLLAALLLLQRSPIAHAFZD8-Fc amino acid sequence-variant 54F03 (withoutpredicted signal sequence; the ″GRA″ linker sequence between the FZD8 sequence and the Fc sequence of the fusion protein is underlined)  (SEQ ID NO: 109)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKFZD8-Fc variants FZD8-Fc variant 54F03 amino acid sequence (withoutpredicted signal sequence; alternative cleavage)  (SEQ ID NO: 110)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKFZD8-Fc variant 54F09 amino acid sequence (withoutpredicted signal sequence)  (SEQ ID NO: 111)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITAAPSPPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKFZD8-Fc variant 54F09 amino acid sequence (withoutpredicted signal sequence; alternative cleavage)  (SEQ ID NO: 112)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKFZD8-Fc variant 54F15 amino acid sequence (withoutpredicted signal sequence)  (SEQ ID NO: 113)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITAAPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKFZD8-Fc variant 54F15 amino acid sequence (withoutpredicted signal sequence; alternative cleavage)  (SEQ ID NO: 114)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKFZD8-Fc variant 54F16, 54F17, 54F18, 54F23, 54F25,54F27, 54F29, 54F31, and 54F34 amino acid sequence (without predicted signal sequence)  (SEQ ID NO: 115)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLTIKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKFZD8-Fc variant 54F16 amino acid sequence (withoutpredicted signal sequence; alternative cleavage) (SEQ ID NO: 116)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLTIKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGFZD8-Fc variant 54F19, 54F20, 54F24, 54F26, 54F28,54F30, 54F32, 54F34 and 54F35 amino acid sequence (without predicted signal sequence)  (SEQ ID NO: 117)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGKFZD8-Fc variant 54F19 amino acid sequence (withoutpredicted signal sequence; alternative cleavage)  (SEQ ID NO: 118)ALAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKFZD8-Fc variant 54F20 amino acid sequence (withoutpredicted signal sequence; alternative cleavage)  (SEQ ID NO: 119)VLAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKFZD8-Fc variant 54F34 amino acid sequence (withoutpredicted signal sequence)  (SEQ ID NO: 120)KELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLITEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKFZD8-Fc variant 54F33 amino acid sequence (withoutpredicted signal sequence) (SEQ ID NO: 121)KELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRIDLTIKSSDKIHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTRNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKh-Wnt1 C-terminal cysteine rich domain  (aa 288-370) (SEQ ID NO: 122):DLVYFEKSPNECTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRIQRVIERCNCTFHWCCHVSCRNCTHTRVLHECLh-Wnt2 C-terminal cysteine rich domain  (aa 267-360) (SEQ ID NO: 123):DLVYFENSPDYCIRDREAGSLGTAGRVCNLTSRGMDSCEVMCCGRGYDTSHVTRMTKCGCKFHWCCAVRCQDCLEALDVHTCKAPKNADWTTATh-Wnt2b C-terminal cysteine rich domain  (aa 298-391) (SEQ ID NO: 124):DLVYFDNSPDYCVLDKAAGSLGTAGRVCSKTSKGTDGCEIMCCGRGYDTTRVTRVTQCECKFHWCCAVRCKECRNTVDVHTCKAPKKAEWLDQTh-Wnt3 C-terminal cysteine rich domain  (aa 273-355) (SEQ ID NO: 125):DLVYYENSPNFCEPNPETGSFGTRDRTCNVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCIFHWCCYVSCQECIRIYDVHTCKh-Wnt3a C-terminal cysteine rich domain  (aa 270-352) (SEQ ID NO: 126):DLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCKh-Wnt7a C-terminal cysteine rich domain  (aa 267-359) (SEQ ID NO: 127):DLVYIEKSPNYCEEDPVIGSVGIQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCKh-Wnt7b C-terminal cysteine rich domain  (aa 267-349) (SEQ ID NO: 128):DLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCKh-Wnt8a C-terminal cysteine rich domain  (aa 248-355) (SEQ ID NO: 129):ELIFLEESPDYCTCNSSLGIYGTEGRECLQNSHNTSRWERRSCGRLCTECGLQVEERKTEVISSCNCKFQWCCTVKCDQCRHVVSKYYCARSPGSAQSLG RVWFGVYIh-Wnt8b C-terminal cysteine rich domain  (aa 245-351) (SEQ ID NO: 130):ELVHLEDSPDYCLENKTLGLLGTEGRECLRRGRALGRWELRSCRRLCGDCGLAVEERRAETVSSCNCKFHWCCAVRCEQCRRRVTKYFCSRAERPRGGAA HKPGRKPh-Wnt10a C-terminal cysteine rich domain  (aa 335-417) (SEQ ID NO: 131):DLVYFEKSPDFCEREPRLDSAGTVGRLCNKSSAGSDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCKh-Wnt10b C-terminal cysteine rich domain  (aa 307-389) (SEQ ID NO: 132):ELVYFEKSPDFCERDPIMGSPGIRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCKFZD8-Fc variant 54F28 with predicted signal  sequence underlined (SEQ ID NO: 133) MEWGYLLEVTSLLAALLLLQRSPFVHAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-6. (canceled)
 7. A method of treating neuroendocrine cancer,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a Wnt antagonist.
 8. The method of claim 7, whereinthe subject is a human subject. 9-10. (canceled)
 11. The method of claim7, wherein the neuroendocrine tumor is selected from the groupconsisting of gastroenteropancreatic neuroendocrine tumor, carcinoidtumor, pheochromocytoma, paraganglioma, medullary thyroid cancer,pulmonary neuroendocrine tumor and thymic neuroendocrine tumor.
 12. Themethod of claim 11, wherein the neuroendocrine tumor is a carcinoidtumor or a pancreatic neuroendocrine tumor.
 13. The method claim 7,wherein the Wnt antagonist is an antibody that binds to at least onehuman frizzled receptor (FZD). 14-16. (canceled)
 17. The method of claim13, wherein the antibody specifically binds to at least one human FZDselected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8.18-21. (canceled)
 22. The method of claim 17, wherein the antibodyspecifically binds to FZD1, FZD2, FZD5, FZD7, and FZD8.
 23. (canceled)24. The method of claim 13, wherein the antibody blocks Wnt binding toFZD or blocks the activation of FZD.
 25. (canceled)
 26. The method ofclaim 13, wherein the antibody comprises: (a) a heavy chain CDR1comprising GFTFSHYTLS (SEQ ID NO:31), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:32), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:33); and/or (b) (i) a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:34), a light chain CDR2 comprising DKSNRPSG (SEQID NO:35), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:36);or (ii) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:41), alight chain CDR2 comprising EKDNRPSG (SEQ ID NO:42), and a light chainCDR3 comprising SSFAGNSLE (SEQ ID NO:43).
 27. The method of claim 26,wherein the antibody comprises: (a) a VH comprising the amino acidsequence of SEQ ID NO:37; and/or (b) a VL comprising the amino acidsequence of SEQ ID NO:38 or
 44. 28. The method of claim 26, wherein theantibody comprises: (a) a heavy chain comprising the amino acid sequenceof SEQ ID NO:39; and/or (b) a light chain comprising the amino acidsequence of SEQ ID NO:40 or
 45. 29. The method of claim 13, wherein theantibody is a monoclonal antibody.
 30. The method of claim 29, whereinthe antibody is a recombinant antibody, a chimeric antibody, a humanizedantibody, a human antibody, or an antibody fragment.
 31. The method ofclaim 29, wherein the antibody is a monospecific antibody or abispecific antibody.
 32. (canceled)
 33. The method of claim 13, whereinthe antibody is an IgG1 or IgG2 antibody.
 34. The method of claim 7,wherein the Wnt antagonist is a soluble FZD receptor that is capable ofbinding to Wnt. 35-36. (canceled)
 37. The method of claim 34, whereinthe soluble receptor comprises a fragment of the extracellular domain ofthe human FZD receptor, said fragment comprising the Fri domain of thehuman FZD receptor.
 38. (canceled)
 39. The method of claim 37, whereinthe human FZD receptor is FZD8.
 40. The method of claim 39, wherein theFZD8 Fri domain comprises the amino acid sequence of SEQ ID NO:
 28. 41.The method of claim 40, wherein the soluble receptor further comprises ahuman Fc domain.
 42. The method of claim 41, wherein the human Fc domaincomprises the amino acid sequence of SEQ ID NO:
 95. 43. The method ofclaim 7, which further comprises administering a second therapeuticagent to the subject.
 44. The method of claim 43, wherein the secondtherapeutic agent is a chemotherapeutic agent. 45-48. (canceled)
 49. Themethod of claim 7, wherein the Wnt antagonist is OMP-18R5. 50-52.(canceled)
 53. The method of claim 7, wherein the Wnt antagonist isOMP-54F28.
 54. The method of claim 7, wherein the neuroendocrine tumoris not small cell lung tumor (SCLC). 55-56. (canceled)